TAGZyme ™ Handbook

For Exoproteolytic cleavage of N-terminal His tags

July 2015 © 2001–2015 QIAGEN, all rights reserved.

www.qiagen.com Contents

Kit Contents 5 Storage and Stability 6 Safety Information 6 Product Use Limitations 7 Product Warranty and Satisfaction Guarantee 7 Technical Assistance 7 Introduction 8 The TAGZyme Principle 9 Proteins with intrinsic DAPase stop points 9 Proteins without intrinsic DAPase stop points 10 Applications for the TAGZyme System 12 TAGZyme pQE Vectors 13 QIA express pQE vectors 13 Regulation of expression – pREP4 plasmid 14 E. coli host strains 15 The Cloning Procedure 17 Cleavage Protocols 20 Notes Before Starting 20 His tag design 20 Expression, Ni-NTA purification, and desalting of His-tagged proteins 20 Buffers and enzyme activity 21 DAPase Enzyme 21 Qcyclase Enzyme 21 pGAPase Enzyme 22 Cysteamine activation 22 Stepwise Use of the TAGZyme System 22 Analysis during the TAGZyme procedure 23 Cleavage of N-terminal 6xHis tags using DAPase Enzyme 25 Buffer preparation 25 Desalting 25 Protocol 1. DAPase digestion (small-scale pilot experiment) for proteins containing intrinsic DAPase stop points 26

TAGZyme Handbook 07/2015 3 Protocol 2. Preparative DAPase digestion for proteins containing intrinsic DAPase stop points 28 Protocol 3. Removal of DAPase Enzyme by subtractive IMAC 29 Cleavage of N-terminal His tags using DAPase, Qcyclase, and pGAPase Enzymes 30 Buffer preparation 30 Desalting 30 Protocol 4. DAPase – Qcyclase digestion (small-scale pilot experiment) 31 Protocol 5. Preparative DAPase – Qcyclase digestion 33 Protocol 6. Removal of DAPase and Qcyclase Enzymes by subtractive IMAC 34 Protocol 7. pGAPase Digestion 35 Protocol 8. Preparative pyroglutamate removal using pGAPase Enzyme 37 Protocol 9. Removal of pGAPase Enzyme by subtractive IMAC 38 Troubleshooting Guide 39 Appendix 41 Enzyme specifications 41 Solutions required 44 Use of blunt-end–cutting restriction enzymes to introduce various amino acids at the N-terminus of the mature protein 44 Recommended PCR cloning strategy for use of the vector pQE-TriSystem in 48 combination with TAGZyme cleavage His tags suitable for exoproteolytic cleavage by the TAGZyme system 50 Small-scale subtractive IMAC 51 Recommendations for subtractive IMAC scale-up 51 References 52 Ordering Information 54 QIAGEN Distributors 55

4 TAGZyme Handbook 07/2015 Kit Contents

TAGZyme ™ Kit — for processing of approx. 10 mg of tagged protein Catalog No. 34300 DAPase ™ Enzyme (10 U/ml) 0.5 units (50 µl) Qcyclase ™ Enzyme (50 U/ml) 30 units (600 µl) pGAPase ™ Enzyme (25 U/ml) 10 units (400 µl) Cysteamine·HCl (20 mM) 1000 µl Ni-NTA Agarose 10 ml Disposable columns (empty) 20 Handbook 1 Product sheets DAPase Enzyme and Qcyclase/pGAPase Enzymes each 1

TAGZyme pQE Vector Set Catalog No. 32932 Vectors TAGZyme pQE-1 and -2 each 25 µg Handbook 1

TAGZyme DAPase Enzyme (2.5 U) — for processing of approx. 50 mg of tagged protein Catalog No. 34362 DAPase Enzyme (10 U/ml) 2.5 units (250 µl) Cysteamine·HCl (20 mM ) 1000 µl Handbook 1 Product sheet DAPase Enzyme 1

TAGZyme Qcyclase/ pGAPase Enzymes (150 U/50 U) — for processing of approx. 50 mg of tagged protein Catalog No. 34342 Qcyclase Enzyme (50 U/ml) 150 units (5 x 600 µl) pGAPase Enzyme (25 U/ml) 50 units (5 x 400 µl) Handbook 1 Product sheet Qcyclase/pGAPase Enzymes 1

TAGZyme Handbook 07/2015 5 TAGZyme DAPase Enzyme (50 U)* — for processing of approx. 1 g of tagged protein Catalog No. 34366 DAPase Enzyme (10 U/ml) 50 units (5 ml) Cysteamine·HCl (20 mM ) 25 ml Handbook 1 Product sheet DAPase Enzyme 1

TAGZyme Qcyclase/pGAPase Enzymes (3000 U/1000 U)* — for processing of approx. 1 g of tagged protein Catalog No. 34346 Qcyclase Enzyme (50 U/ml) 3000 units (5 x 12.5 ml) pGAPase Enzyme (25 U/ml) 1000 units (5 x 8 ml) Handbook 1 Product sheet Qcyclase/pGAPase Enzymes 1

* Bulk enzyme quantities are customized products; delivery may take up to 6 weeks. Enzymes are also avail able in GMP-grade. Please inquire. Storage and Stability DAPase ™, Qcyclase ™, and pGAPase ™ Enzymes, Cysteamine·HCl, and vectors TAGZyme pQE-1 and -2 should be stored at –30 to –15 °C. Ni-NTA Agarose should be stored at 2–8°C. Ni-NTA matrices should not be frozen. Disposable columns can be stored at room temperature. See Appendix for enzyme stability information.

Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component.

6 TAGZyme Handbook 07/2015 Product Use Limitations The TAGZyme ™ System is developed, designed, and sold for research purposes only. It is not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use. All due care and attention should be exercised in the handling of many of the materials described in this text.

Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product — as you wish. A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see inside front cover).

Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products. If you have any questions or experience any difficulties regarding the TAGZyme System or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors (see inside front cover).

TAGZyme Handbook 07/2015 7 Introduction The TAGZyme System is an efficient and specific solution for the complete removal of small N-terminal His tags and other amino acid tags by the use of exopeptidases. The method is based on the use of dipeptidyl aminopeptidase I (DAPase Enzyme) alone or in combination with glutamine cyclotransferase (Qcyclase Enzyme) and pyroglutamyl aminopeptidase (pGAPase Enzyme). These recombinant enzymes contain a C-terminal His tag and can therefore be bound to Ni-NTA matrices. This allows their removal from the reaction solution by Immobilized-Metal Affinity Chromatography (IMAC). This feature has been utilized in the design of a simple process consisting of aminopeptidase cleavage followed by subtractive IMAC. The system can form the backbone of a combined TAGZyme–IMAC strategy for the efficient production of highly purified and homogeneous recombinant proteins (see Figure 1). As the TAGZyme enzymes carry a His tag at their C-terminus, the tag is not digested by the N-terminal exopeptidase DAPase Enzyme. The use of exopeptidases in the TAGZyme System eliminates the risk of endoproteolytic cleavage within the body of the protein, and the rapid rate of reaction means that sensitive proteins can be processed even at 4°C in a matter of hours.

Cr ude His-tagged target protei n

IMAC

Purified His-tagged target protei n

Tag digestion

TAGZyme protei n Subtractive IMAC

Target protein

Figure 1. Schematic summary of the TAGZyme procedure.

8 TAGZyme Handbook 07/2015 The TAGZyme Principle The TAGZyme System makes use of a highly specific and processive exoproteolytic cleavage of N-terminal amino acids for the removal of small affinity tags from proteins (e.g., 6xHis tags). After completion of the digestion, the exoproteolytic enzymes — which carry His tags at the C-terminus — are removed by subtractive IMAC. The efficiency of the total procedure is supplemented by the use of a series of E. coli expression vectors, TAGZyme pQE-1 and -2. These expression vectors combine the high-level expression of 6xHis-tagged proteins delivered by the pQE vector series with specially designed His tag-coding and multiple cloning site sequences that allow complete and convenient removal of N-terminal His tags. The two vectors have been optimized for use with the TAGZyme System. Occasionally, problems may be encountered when processing other tags. For an overview of the suitability of other tags for TAGZyme processing, please consult Table 2 (page 17). N-terminal amino acids are cleaved off as dipeptides by dipeptidyl aminopeptidase I (DAPase Enzyme, Figure 2A). After removal of the first N-terminal dipeptide the enzyme progressively cleaves off dipeptides until a stop point is encountered. Stop points are certain amino acids in defined positions within a dipeptide (see Table 1). As the His tags of TAGZyme enzymes appear at the C-terminus they are not subject to digestion.

Table 1. DAPase stop points

Amino acid DAPase stop point ( ↓) sequence* Lysine (Lys, K) Xaa-Xaa…Xaa-Xaa ↓ Lys -Xaa Xaa-Xaa... Arginine (Arg, R) Xaa-Xaa…Xaa-Xaa ↓ Arg -Xaa Xaa-Xaa... Proline (Pro, P) Xaa-Xaa…Xaa-Xaa ↓ Xaa-Xaa Pro -Xaa... Proline (Pro, P) Xaa-Xaa…Xaa-Xaa ↓ Xaa-Pro Xaa-Xaa... † Glutamine (Gln, Q) Xaa-Xaa…Xaa-Xaa ↓ Gln -Xaa Xaa-Xaa...

* Natural DAPase stop points ( ↓) are the following amino acids in the given position within a dipeptide (dipeptides that are cleaved off are underlined). † In the presence of excess Qcyclase Enzyme.

Proteins with intrinsic DAPase stop points A range of mature proteins have natural DAPase Enzyme stop points (Table 1) causing the enzyme to release its substrate, i.e., the detagged mature protein, as soon as this amino acid occurs in the N-terminal position. Accordingly, N-terminal His tags containing an even number of amino acid residues can be completely and specifically removed by treatment with DAPase Enzyme alone (Figure 2A). After digestion is complete, the C-terminally His-tagged DAPase Enzyme, residual undigested target protein, and copurified contaminants are removed by subtractive IMAC . The entire process is typical ly completed within 45 minutes.

TAGZyme Handbook 07/2015 9 Proteins without intrinsic DAPase stop points cDNA coding for proteins that do not contain a natural stop point can be cloned into a TAGZyme pQE vector in such a way that a glutamine (Gln, Q) residue is introduced into the position between the last cleavable dipeptide and the first authentic amino acid of the target protein. DAPase cleavage is then performed in the presence of a second enzyme, glutamine cyclotransferase (Qcyclase Enzyme), which catalyzes the cyclization of an N-terminal glutamine residue to pyroglutamate (Figure 2B). Presence of excess Qcyclase Enzyme ensures immediate cyclization of the inserted glutamine residue when the His tag has been digested and the glutamine appears in the N-terminal position. A protein possessing an N-terminal pyroglutamate residue cannot serve as a substrate for DAPase Enzyme and is thus protected against further DAPase digestion. Following the coincubation, both enzymes are removed from the reaction solution by subtractive Ni-NTA IMAC. The target protein is obtained by cleaving off the N-terminal pyroglutamyl residue through the action of a third enzyme, pyroglutamyl aminopeptidase (pGAPase Enzyme) (Figures 2B). This process is typically completed within 120 minutes. A glutamine DAPase stop point can be introduced by cloning the protein coding sequence into the expression vector TAGZyme pQE-2. Here, any uneven amino acid position can be chosen for the glu - tamine residue, and the first amino acid of the target protein must immediately follow. Alternatively, TAGZyme pQE-1 is the vector of choice whenever the sequence of the pro - tein allows cloning into the blunt-ended Pvu II restriction site, which links the first amino acid of the target protein to the glutamine stop point (Figure 4, page 16).

10 TAGZyme Handbook 07/2015 Proteins with Intrinsic DAPase Stop Points Proteins without Intrinsic DAPase Stop Points

A B

(Met-Lys)-(His) 2-(His) 2-(His) 2- Stop- Protein (Met-L ys)-(His) 2-(His) 2-(His) 2- GlnStop- Protein

(His) 2-(His) 2-(His) 2- Stop- Protein (His) 2-(His) 2-(His) 2- GlnStop- Protein

DAPase DAPase treatment (His) 2-(His) 2- Stop- Protein (His) 2-(His) 2- GlnStop- Protein treatment

(His) 2- Stop- Protein (His) 2- GlnStop- Protein

Stop- Protein GlnStop- Protein

Qcyclase treatment

pyroGlu- Protein

Subtractive IMAC

Subtractive IMAC

DAPase

TA G Protein DAPase, Qcyclase

pyroGlu- Protein TA G ~ 45 mi n pGAPase treatment

Subtractive IMAC

pGAPase

Protein

~ 120 min Figure 2. Combined cleavage and purification strategy. IA Procedure for proteins having an intrinsic DAPase stop point IB Procedures for proteins with an introduced glutamine DAPase stop point.

TAGZyme Handbook 07/2015 11 Applications for the TAGZyme System The TAGZyme System is suitable for cleavage of every N-terminal His tag that contains certain sequence characteristics that are described in this handbook.

Therapeutic proteins In the production of therapeutic proteins for use in humans or animals, it is often desirable to remove any artificial additions such as His tags. The specificity and the speed of action of the recombinant enzymes in the TAGZyme System make it an ideal tool for production of recombinant proteinaceous therapeutics, where precisely defined cleavage characteristics are required. Due to the independence of this system from sequence-determined cleavage sites, the TAGZyme System provides the highest level of specificity of protein cleavage and target protein integrity. TAGZyme System components are available in various formats including bulk quantities of single enzymes. Moreover, the TAGZyme System can be combined with Ni-NTA–based IMAC protein purification strategies for which Drug Master Files (Type II) are available for support in regulatory affairs. The subtractive Ni-NTA IMAC processing steps within the TAGZyme procedure can contribute to an increase in the purity of the target protein. For production where the highest safety standards are required, GMP-quality TAGZyme enzymes are available. Please contact QIAGEN for details (see inside front cover).

Protein crystallography and structure determination Structure determination of protein crystals has become more and more important in the context of drug screening and rational design of small molecules for protein ligands. In order to elucidate a protein’s structure it is often desirable to crystallize the protein in the native state, i. e., without affinity tag additions. Screening for crystallization conditions is often done in a high-throughput format. QIAGEN offers an automated solution for the production of large amounts of His-tagged protein for this purpose in 96-well format. TAGZyme is an ideal supplementary tool for this automated protein purification. Please contact QIAGEN for further details.

Protein microarrays Depending on the chemistry of coupling of a purified protein to the surface of a chip and on the chip’s intended application, it may be desirable to remove His tags from proteins prior to immobilization. In combination with an automated solution for the purification of His-tagged proteins in a high-throughput format, TAGZyme is the method of choice to produce a large number of authentic, detagged proteins in a convenient and timesaving procedure. Please contact QIAGEN for details of available solutions in automated high-throughput protein purification and downstream processing.

12 TAGZyme Handbook 07/2015 Basic research Because the TAGZyme System is available in a small kit size and protocols are provided for the processing of a large range of proteins, the system is equally suited to His-tag removal from proteins used in small assays in basic research.

TAGZyme pQE Vectors The vectors TAGZyme pQE-1 and -2 are based on the pQE-30 and pQE-80 vectors respectively, and are used for expression of N-terminally 6xHis-tagged proteins in E. coli .

The His tag has been modified to encode the epitope Met-Lys-His 6 (MKH 6) a short His tag and the N-terminus Met-Lys motif that delivers a high expression level and lowest level of methionine processing in E. coli (1). Furthermore, in the rare case of methionine process ing in E. coli the N-terminal lysine residue would act as a DAPase stop point (see Table 1, page 9) and prevent DAPase from out-of-frame digestion. The presence of this DAPase stop point leaves the protein intact allowing removal of the protein by subtractive IMAC.

QIA express pQE vectors High-level expression of 6xHis-tagged proteins in E. coli using the QIA express pQE vectors is based on the T5 promoter transcription-translation system. pQE plasmids belong to the pDS family of plasmids (2) and were derived from plasmids pDS56/RBSII and pDS781/RBSII-DHFRS (3). These low-copy plasmids (Figure 3) have the following features: • Optimized promoter-operator element consisting of phage T5 promoter (recognized by the E. coli RNA polymerase) and two lac operator sequences which increase lac repressor binding and ensure efficient repression of the powerful T5 promoter • Synthetic ribosomal binding site, RBSII, for high translation rates • 6xHis-tag coding sequence either 5' or 3' to the cloning region • Multiple cloning site and translational stop codons in all reading frames for convenient preparation of expression constructs

• Two strong transcriptional terminators: t 0 from phage lambda (4), and T1 from the rrnB operon of E. coli , to prevent read-through transcription and ensure stability of the expression construct

• β-lactamase gene ( bla ) confers resistance to ampicillin (5) at 100 µg/ml (the chloramphenicol acetyl transferase gene (CAT) present between t 0 and T1 has no promoter and is not normally expressed) • ColE1 origin of replication (5) Restriction maps and sequences for the cloning regions of QIA express vectors are presented in Figure 5. The entire sequence information is available at www.qiagen.com. A strategy for the use of the pQE-TriSystem vector for protein expression in insect and mammalian cells in combination with the TAGZyme System is outlined in the Appendix, page 48.

TAGZyme Handbook 07/2015 13 Xho I (0/3416) promoter/operator

bla Eco RI

RBS II Bgl I (2544) pQE (121) polylinker/ 6xHis tag

t0 T1 ori Xba I (1118)

Figure 3. pQE vectors.

Regulation of expression — pREP4 plasmid The extremely high transcription rate initiated at the T5 promoter can only be efficiently regulated and repressed by the presence of high levels of the lac repressor protein. E. coli host strains used in the QIA express System use a lac repressor gene in trans or cis to the gene to be expressed. In the trans system, the host strains contain the low-copy plasmid pREP4 which confers kanamycin resistance and constitutively expresses the lac repressor protein encoded by the lac I gene (6). The pREP4 plasmid is derived from pACYC and contains the p15A replicon. Multiple copies of pREP4 are present in the host cells that ensure the production of high levels of the lac repressor protein that binds to the operator sequences and tightly regulates recombinant protein expression. The pREP4 plasmid is compatible with all plasmids carrying the ColE1 origin of replication, and is maintained in E. coli in the presence of kanamycin at a concentration of 25 µg/ml. The cis -repressed vectors pQE-80L, -81L, and -82L contain the lac l q gene and do not require the presence of pREP4. Expression of recombinant proteins encoded by pQE vectors is rapidly induced by the addition of isopropyl- β-D-thiogalactoside (IPTG) which binds to the lac repressor protein and inactivates it. Once the lac repressor is inactivated, the host cell’s RNA polymerase can transcribe the sequences downstream from the promoter. The transcripts produced are then translated into the recombinant protein. The special “double operator” system in the pQE expression vectors, in combination with the high levels of the lac repressor protein generated by pREP4 or the lacl q gene on pQE-80L, pQE-81L, or pQE-82L, ensure tight control at the transcriptional level. The pREP4 plasmid is already present in the QIA express E. coli strains M15[pREP4] and SG13009[pREP4]. Using pQE-80L, pQE-81L, or pQE-82L with the cis- lacl q gene, expression rates are comparable with those obtained using pQE-30, pQE-31, or pQE-32 vectors in combination with pREP4.

14 TAGZyme Handbook 07/2015 E. coli host strains Any E. coli host strain containing both the expression (pQE) and the repressor (pREP4) plasmids can be used for the production of recombinant proteins. The QIA express System uses E. coli strain M15[pREP4] which permits high-level expression and is easy to handle. Strain SG13009[pREP4] (7) is also supplied and may be useful for the production of proteins that are poorly expressed in M15[pREP4]. Both the M15 and SG13009 strains are derived from E. coli K12 and have the phenotype NaI s, Str s, Rif s, Thi -, Lac -, Ara +, Gal +, Mtl -, F -, RecA +, Uvr +, Lon +. E. coli strains that harbor the lacI q mutation, such as XL1 Blue, JM109, and TG1, produce enough lac repressor to efficiently block transcription, and are ideal for storing and propagating pQE plasmids. These strains can also be used as expression hosts for expressing nontoxic proteins, but they may be less efficient than the M15[pREP4] strain, and expression is regulated less tightly than in strains harboring the pREP4 plasmid. If the expressed protein is toxic to the cell, “leaky” expression before induction may result in poor culture growth or in the selection of deletion mutants that grow faster than bacteria containing the correct plasmid. Note that E. coli strains M15 and SG13009 do not harbor a chromosomal copy of the lacI q mutation, so pREP4 must be maintained by selection for kanamycin resistance. The cis -repressed pQE-80L series of vectors can be easily used with any E. coli host strain and kanamycin selection is not necessary.

TAGZyme Handbook 07/2015 15 A PvuII Sph I Sac I No tI Sac II Kpn I Pm lI Sal I Pst I Hi ndIII CAG CTG CAT GCG GGA GCT CAT GCG GCC GCG GGT ACC CAC GTG TCG ACC TGC AGA AGC TT MK HH HH HH ↓ Q stop I I I I I I I I I I I d I I t l I n c h c l n u t o i p a p a a m v s S K P P S S N P H S

PT5 lac O lac O RBS AT G 6xHis MCS Stop Codons

n i l l i c i p TA GZyme m A pQE-1 3.5 kb

Col E1

B Nd eI Sph I SacI No tI Sac II Kpn I Pm lI Sal I Pst I Hi ndIII CAT ATG CAT GCG GGA GCT CAT GCG GCC GCG GGT ACC CAC GTG TCG ACC TGC AGC CAA GCT T MK HH HH HH HM HA GA H AAA GT HV ST CS QA I I I I I I I I I I I d I e t l I n h c c l n t o d i p p a a a s m P S S N S K S P H N

PT5 lac O lac O RBS AT G 6xHis MCS Stop Codons

n i l l i c i

p

m TA GZyme

A

q pQE-2 I

c

a 4.8 kb l

Col E1

Figure 4. TAGZyme pQE vectors for N-terminal His tag constructs which facilitate exoproteolytic removal of N-terminal residues by TAGZyme enzymes. Restriction sites within the multiple cloning sites, DNA sequences, and the corresponding N-terminal amino acid sequences are shown. IA DAPase cleaves off the underlined dipeptides and DAPase digestion stops at the glutamine residue (Q) at position “ ↓” in the presence of excess Qcyclase. IB DAPase Enzyme cleaves off the underlined dipeptides until an introduced stop point is reached.

Refer to The QIA expressionist (8) or to www.qiagen.com for further details and sequences of the TAGZyme pQE-1 and -2 vectors.

16 TAGZyme Handbook 07/2015 The Cloning Procedure The cleavage activity of DAPase Enzyme is dependent upon the amino acid composition of the His tag to be digested, i.e., of the dipeptides to be cleaved. The TAGZyme pQE E. coli expression vectors have been specially designed to provide reliable and efficient His tag cleavage. The TAGZyme pQE-2 tag sequences allow removal of the complete N-terminal tag irrespective of the cloning site of the DNA insert. Vector TAGZyme pQE-1 can be used to insert a glutamine DAPase stop point. See Table 13 in the Appendix for a list of blunt-cutting restriction enzymes that can be used in conjunction with Pvu II for PCR cloning of expression inserts. After IMAC purification and subsequent treatment with DAPase Enzyme and excess Qcyclase Enzyme, the pyroglutamate residue generated can then be removed by the use of pGAPase Enzyme (see above).

Table 2. Examples of the cleavage rates of various dipeptides by DAPase Enzyme.

Rapid rate Medium rate Slow rate No cleavage = of cleavage of cleavage of cleavage DAPase stop point Xaa – Arg Xaa – Asp* Xaa – Phe † Arg – Xaa Xaa – Lys Xaa – Glu* Gly – Met Lys – Xaa His – Ala Phe – Xaa † Gly – Ser Xaa – Pro His – Gln Ala – Ala Ser – Met Xaa – Xbb – Pro His – Gly Asp – Asp*, ‡ Gln – Xaa § His – His Glu – Glu*, ‡ His – Met Glu – His* Ala – His Gly – Phe † Gly – His Ser – Tyr Met – His Ser – Thr His – Val Gly – Ala Gly – Thr

* Positively or negatively charged side chains inhibit DAPase Enzyme cleavage, and sequences containing aspartic acid (Asp) or glutamic acid (Glu) can only be digested by DAPase Enzyme at acidic pH while sequences containing histidine (His) residues require a pH above 6.0. Therefore, 6xHis tags containing Glu or Asp can only be digested by DAPase Enzyme at a pH between 6.0 and 6.5. † With a few exceptions, dipeptides containing Phe, Ile, Leu, Tyr, and Trp in either of the two positions of the dipeptide, are subject to slow cleavage. ‡ Medium to slow cleavage rate. § Gln-Xaa serves as a stop point for DAPase Enzyme only in the presence of Qcyclase Enzyme, which converts the glutamine residue to pyroglutamate (which can be removed by the action of pGAPase Enzyme).

TAGZyme Handbook 07/2015 17 The use of expression vectors other than TAGZyme pQE constructs in conjunction with TAGZyme enzymes is possible. However, since these vectors have not been specifically designed for use with the TAGZyme System, the amino acid sequence to be cleaved off should be carefully analyzed for efficiency of dipeptide cleavage using Table 2. It is also recommended to make use of Table 2 and the guidelines given below whenever a new N-terminal tag is designed for cleavage by DAPase Enzyme or in combination with Qcyclase Enzyme and pGAPase Enzyme. The TAGZyme System is equally suited for use with prokaryotic and eukaryotic expression systems. In addition to considering the dipeptide composition within the N-terminus to be cleaved, the following guidelines should be followed in order to achieve successful removal of His tags from recombinant proteins using the TAGZyme System.

• The total number of amino acid residues in the tag to be cleaved off should not exceed 30. • If the target protein contains an intrinsic DAPase stop point (see Table 1) which is to be utilized, the tag must be composed of an even number of residues. If the target protein does not contain a stop point the tag must be C-terminally extended with a glutamine residue (Q) in addition to an even number of residues N-terminal to the glutamine residue. • Arginine (R) and lysine (K) residues must not be placed in odd-numbered positions of the tag. • The tag sequence must not contain any proline (P) residues. • The N-terminal sequence of the tag should be designed to minimize in vivo processing of the N-terminal methionine residue. Processing of methionine leads to out-of-frame DAPase digestion and results in the accumulation of erroneously processed products. In E. coli , the residue adjacent to the initial methionine influences the degree of processing, and a bulky residue should be placed in this position. We recommend lysine (found in TAGZyme pQE-1 and -2 vectors), but arginine, glutamate, or aspartate are also effective as a penultimate residue that reduces methionine processing in E. coli (1). Please note that DAPase–Qcyclase Enzyme treatment must be carried out at pH 6.0–6.5 if aspartate or glutamate residues occur in the His-tag sequence to be cleaved (see Table 2 and Table 3).

18 TAGZyme Handbook 07/2015 Table 3. Characteristics of recommended His tag N-termini

Level of methionine Expression DAPase Enzyme processing level in cleavage Other N-terminus in E. coli * E. col i† rate advantages Met 1 Lys 2 – His tag Very low Very high Rapid DAPase Enzyme stop point for out-of-frame digestion ‡ Met 1 Arg 2 – His tag Low Very high Rapid DAPase Enzyme stop point for out-of-frame digestion ‡ Met 1 Glu 2 – His tag Low High Medium (only at acidic pH) Met 1 Asp 2 – His tag Low High Medium (only at acidic pH)

* See reference 1 and 11. † See reference 1. ‡ See also Table 1.

In general, the nucleotide sequence encoding the tag has a strong influence on the expression level and self-complimentary mRNA sequences close to the ATG start codon should be avoided. Furthermore, it has been observed that the amino acids lysine (codon AAA) and arginine (example codon CGT) significantly improve expression if placed next to the start methionine coded by ATG. The tag sequence should be designed to avoid dipeptides that result in slow DAPase cleavage rates. The rate at which DAPase Enzyme cleaves off different dipeptides from the N-terminus depends on the amino acid residues in the dipeptide. Dipeptides containing an arginine or lysine residue in the C-terminal position are cleaved off very quickly, whereas dipeptides containing a hydrophobic amino acid residue (i.e, Phe, Ile, Leu, Tyr, Trp) are cleaved off relatively slowly. Examples of cleavage rates with respect to dipeptide composition are shown in Table 2, page 17. Proteins expressed using other pQE vectors that encode N-terminal His tags are poorly processed as they encode the sequence Met-Arg-Gly-Ser. The second dipeptide, Gly-Ser is slowly cleaved by DAPase Enzyme (see Table 2). Furthermore, C-terminal His tags encoded by the pQE vectors pQE-60, -70, -16, and -TriSystem are not removed by the TAGZyme System, as the exoprotease cleaves dipeptides from the N-terminus of proteins.

TAGZyme Handbook 07/2015 19 In some cases, in vivo His-tag cleavage can occur, leading to erroneously processed products. Such processing may be observed when arginine residues appear in the middle of His-tag sequence or close to the target protein sequence. It is recommended that arginine residues only appear close to the N-terminus of the tag. In response to out-of-frame digestion resulting from methionine processing or in vivo cleavage of the tag, incorporation of DAPase stop points in the tag is recommended (see above). Erroneously processed products with two or more consecutive histidine residues remaining can be efficiently removed by subtractive Ni-NTA IMAC after DAPase digestion. Lysine, arginine, and alternating glutamine residues (in the presence of Qcyclase Enzyme) are functional stop points. It is recommended to place arginine in the N-terminal sequence of a tag (see above).

Cleavage Protocols

Notes Before Starting

His-tag design Please note that the precise amino acid sequence of a polyhistidine tag and the nucleotide sequence selected to encode it are of great importance for the overall performance of the resulting construct during expression, post-translational processing, purification, and tag removal. For these reasons, we strongly recommend using the pQE-TAGZyme expression vectors that have been optimized for use with the TAGZyme System. If other vectors are used, please follow the guidelines given in this handbook regarding tag design and gene construction strategy.

Expression, Ni-NTA purification, and desalting of His-tagged proteins For expression and purification of 6xHis-tagged proteins refer to The QIA expressionist, a handbook for high-level expression and purification of 6xHis-tagged proteins (8). The TAGZyme procedure works well at pH 6.3–7.5 in the absence of imidazole. Because an addition of imidazole or a reduction in pH are usually used for elution of His-tagged proteins from Ni-NTA matrices, the purified target should be desalted before starting the TAGZyme procedure. Desalting can be performed using standard techniques such as gel filtration, ultrafiltration, hollow-fibre modules, or dialysis. The presence of imidazole leads to a decrease in the cleavage rate, probably due to competitive inhibitory effect on DAPase catalysis. Simple dilution of IMAC elution fractions for direct use in the TAGZyme procedure without prior desalting may be possible but reaction parameters must be optimized in pilot experiments. Imidazole should be diluted to a concentration of 50 mM or lower. The final protein concentration should not be lower than 0.3 mg/ml.

20 TAGZyme Handbook 07/2015 Buffers and enzyme activity Phosphate buffers are recommended for use with the TAGZyme System due to their low tendency to strip metal ions from IMAC matrices. However, other buffers may be used such as Tris, Bis-Tris, Bis-Tris·Propane, and MES. For further details on the compatibility of reagents with Ni-NTA IMAC matrices and procedures, refer to Table 4 in The QIA expressionist (8) or visit www.qiagen.com. The optimal pH for TAGZyme catalyzed removal of tags, and also for removal of His-tagged contaminants using subtractive IMAC, is 6.7 to 7.5. All the TAGZyme enzymes work well between 4°C and 37°C as outlined in Table 4.

Table 4. Effect of reaction temperature on enzymatic activity

Reaction temperature Relative activity of TAGZyme enzymes 37°C 100% Room temperature (20–25°C) 50% 4–6°C 10%

DAPase Enzyme DAPase Enzyme is inactive in the presence of denaturants (guanidine hydrochloride or urea), even at low concentrations. DAPase Enzyme is 85–100% active in the presence of up to 0.4% CHAPS, Tween ® 20, Tween 80, and Triton ® X-100 and 60–70% active in the presence of 1% of these detergents. The enzyme has a broad pH optimum between pH 4 and 8. However, the activity depends on the dipeptides to be cleaved off (see Table 2 and Table 3). The optimal pH of DAPase-catalyzed removal of His tags is pH 6.7 to 7.2. The enzyme’s activity is approximately 50% of maximum at pH 6.3 and 7.5 and approximately 10% at pH 5.8 and 8.0. DAPase Enzyme must be activated before use by the reducing agent cysteamine (see protocols).

Qcyclase Enzyme Qcyclase Enzyme has a pH optimum of 8 and shows approximately 60–70% of maximum activity at pH 7 and pH 9.5 and approximately 40% at pH 6.2. Qcyclase Enzyme is inactive below pH 4.5. Qcyclase Enzyme is not inactivated by reducing agents such as cysteamine and DTT.

TAGZyme Handbook 07/2015 21 pGAPase Enzyme pGAPase Enzyme is 100% stable in the presence of 1.6 M guanidine hydrochloride for 140 minutes and shows approximately 85% of maximum activity in the presence of 3.2 M guanidine hydrochloride for 140 minutes. Because pGAPase Enzyme is a cysteine peptidase, urea is not recommended for use with pGAPase Enzyme because the presence of low amounts of the chemical decomposition product cyanate will inactivate the enzyme. The enzyme is 85–100% active in the presence of up to 0.4% CHAPS, Tween 20, Tween 80, and Triton X-100 and about 60% active in the presence of 1% of these detergents. pGAPase Enzyme is slightly activated by sarkosyl and shows 125% activity in the presence of 1% sarkosyl. pGAPase Enzyme has a broad pH optimum between pH 6 and 10 with approximately 70% activity at pH 6 and 10. pGAPase Enzyme must be activated before use by the reducing agent cysteamine (see protocols). TAGZyme Buffer (see below) provides optimal TAGZyme enzyme activity conditions and its use is recommended in TAGZyme procedures.

Cysteamine activation Cysteamine is used as an activator for DAPase Enzyme and pGAPase Enzyme. It is thought that at a physiological pH, cysteamine, and its oxidized form cystamine, act as a hydrogen donor for the reduction of disulfides in the enzymes.

Stepwise Use of the TAGZyme System Normally, by following the recommendations below, sufficient cleavage of the target protein will be obtained. However, the actual rate of cleavage of an N-terminal His tag may vary from protein to protein. This can be due to variations in steric accessibility of the tag or due to modifications of amino acid residues. If the optimal cleavage conditions have to be determined for a specific purification process, it is recommended that a pilot DAPase or DAPase–Qcyclase digestion reaction be carried out.

Therefore, the use of the TAGZyme System should include the following steps: • Pilot digestion to establish optimum enzyme concentrations • Preparative DAPase or DAPase–Qcyclase digestion • Subtractive IMAC using Ni-NTA resin • Digestion with pGAPase Enzyme and second subtractive IMAC (for proteins without intrinsic DAPase stop points)

22 TAGZyme Handbook 07/2015 Analysis during the TAGZyme procedure We recommend that the efficiency of cleavage reactions is checked by subjecting samples from the different steps of the TAGZyme procedure to SDS-PAGE analysis. The pGAPase Enzyme-catalyzed removal of pyroglutamate cannot be observed directly but may be confirmed by subjecting an aliquot of the fully processed protein to a second round of DAPase treatment and subsequent analysis by SDS-PAGE. The result of efficient pGAPase Enzyme-catalyzed pyroglutamate removal will be quantitative DAPase Enzyme-catalyzed conversion of the deprotected target protein (i.e., without an N-terminal pyroglutamate residue) into truncated forms with increased electrophoretic mobility. Figure 5, page 24 shows an SDS-PAGE analysis of a TAGZyme-cleaved His-tagged protein. His-tagged human tumor necrosis factor α (hTNF α) expressed in E. coli and purified by Ni-NTA IMAC, was subjected successively to DAPase–Qcyclase and pGAPase treatment and the authentic target protein hTNF α was recovered from the reaction solution by subtractive IMAC (lane 5). In addition to the TAGZyme enzymes and residual, unprocessed His-tagged TNF α, minor contaminants were efficiently removed in this step. The completeness of the pGAPase Enzyme-catalyzed removal of the pyroglutamate residue was checked by incubation of an aliquot of the fully processed TNF α with DAPase Enzyme. A quantitative DAPase Enzyme-catalyzed conversion of deprotected TNF α into a truncated form with increased electrophoretic mobility was observed (lane 6) confirming efficient pGAPase Enzyme-catalyzed removal of the pyroglutamate residue.

TAGZyme Handbook 07/2015 23 kDa M123456

66.3 – 55.4 –

36.5 – 31.0 – – His-tagged DAPase Enzyme 21.5 – – His-tagged hTNF α – hTNF α 14.4 – – His-tagged DAPase Enzyme 6.0 –

Figure 5. Removal of the His tag from His-tagged hTNF α using the TAGZyme System. 1: Purified His-tagged hTNF α (the tag was an alternating tag as described in the Appendix, “His tags suitable for exoproteolytic cleavage by the TAGZyme System”). 2: After incubation for 10 minutes with DAPase and Qcyclase Enzymes at 37°C. 3: After incubation for 20 minutes with DAPase and Qcyclase Enzymes at 37°C. 4: After incubation for 30 minutes with DAPase and Qcyclase Enzymes at 37°C. 5: After completion of the reaction untagged, pyroglutamyl-extended hTNF α was recovered by subtractive IMAC, subjected to pGAPase digestion, and mature hTNF α was recovered in the flow-through fraction of a second round of subtractive IMAC. 6: An aliquot of the processed protein was incubated with excess DAPase (0.125 U/mg hTNF α) for 2 hours in order to analyze the efficiency of the pGAPase-catalyzed removal of pyroglutamate. The two subunits of His-tagged DAPase Enzyme are visible. All samples were subjected to SDS-PAGE and the gel stained by Coomassie ® Blue. M: markers.

24 TAGZyme Handbook 07/2015 Cleavage of N-terminal 6xHis tags using DAPase Enzyme The data in Table 5 can be used as a rough guide to the amount of DAPase Enzyme necessary to completely digest the N-terminal tag of a recombinant protein. However, substantial differences in activity may be encountered from protein to protein (see above). Therefore, the figures in Table 5 should be considered as a starting point for the pilot experiments only, and not as a general rule for any protein and reaction scale.

Table 5. Times generally required for DAPase digestion at the given DAPase Enzyme-to- target protein ratio

Amount of target Amount of DAPase Reaction Required protein Enzyme temperature reaction time 4°C 5 h 1 mg 50 mU 20–25°C 60 min 50 µg 2.5 mU 37°C 30 min

The following section is for DAPase digestion of tagged proteins containing an intrinsic stop point (see above). For the combined DAPase–Qcyclase and pGAPase digestion see Protocols 4 and 5.

Buffer preparation From a 10x stock solution (see Appendix) prepare enough 1x TAGZyme Buffer (20 mM sodium phosphate, pH 7.0; 150 mM NaCl) for desalting and the TAGZyme digestion. Approximately 50 ml 1x TAGZyme Buffer is sufficient for a small-scale experiment. Note that tags containing glutamate or aspartate residues can only be efficiently cleaved at pH 6.0–6.5. In this case, prepare the TAGZyme Buffer at lower pH (20 mM sodium phosphate, pH 6.0–6.5; 150 mM NaCl).

Desalting Desalt your His-tagged protein using gel filtration, ultrafiltration dialysis, hollow-fibre filtration, or the method best suited to your needs. It is recommended that proteins are exchanged into 1x TAGZyme Buffer. Ni-NTA matrices show negligible leaching of nickel ions into the elution buffer (10). However, when using IMAC resins that bind metals more weakly, addition of 5–10 mM EDTA to the eluted protein fraction prior to the desalting procedure is recommended to remove nickel ions from the His tag.

TAGZyme Handbook 07/2015 25 Protocol 26 A 5 It * T fo r Pr the b d f p d rea Ta a tem or he e ig er ig t D lte rna i µ D E C ble s least i ot lu est est c de d nzy l p AP ys m re am o enz tion t sa ige e er pr g ter oc io io te 6. co a a a mple me tive n a p n n stion yme 0. se tur ot min mme Rec un t vo a m rot ol is liquo /re 3 of ly , e Enz ine lume ei ein s co ed , omm m of can 1 of age nde the from t ns ·H g/ml an mplete of yme or . D in but 5 s Cl the d A D be nt d 0 e r tag pilot co n in equire ndat Pa se each . ( AP that the DAPase µg ( lo 2 1 varie d. The ad after addi mM depen U/ml)* target optim ions expe ta in as e th En zy me reacti re longer samples e E )* tion treatmen nzyme a progress for ri c d al protei in ment tio di ge st i on on co DAPas to g n an inc an d eve nditi should s. d in tr i onto n the varyi t ubat For cyst for Tab in of ry th e e ons 2.5 ea t T on S ea arg 1 E processing 30 io ng AGZ le ns ic a mi nz ta 0 le ng ta n n for ch ne mU rti 6 e ke yme min minut tim the (s m t 5 SD ng stoc gi d yme t th he p p µl ig es (2 utes, DA ves ro S la k poin of d enz yme est es .5 pro s al ce iges tei due ol g b i of g u ion nc ub el a uffer µ Pa n ti l- s add t ces uid in t ons l) tio 50 t 37 an o i nc a be ca le se el si . n ac a th d ° µg SD n vo P in lu t C p c cordi io e ro g an TAGZy me o d e il i lu st S n t w d H ot nc s of in t s al ei me i ng i it t op sam f l g s-t pi lo t im uti en o c ex n yze h a on r to a 0 co 5 e o a Hi tr of pe t g h pl .5 ste n 0 c n at d po in t r nc e ged s en e r 5 Ti e ps of - i – m Handbo ok by im t on u qu 0 a c 5 e tr e t b n U on rat 1 g n t at e 1 u d he , i t xp er im mU t S t ge o r and n D ar ffe ra ig – i ed d i DS o ts o t 10 A 3 h it s reacta d get t es n r n i ions e 00 2 Pas ( on -PAGE. fo r . 0 to pr r ted of an µ . General incub pr 5–5 l ote sho e inactivate µl. co mp l pro 07/2015 ge r ote nts. Enzyme eq control. in en t) toco Lar uld µ u i ation T n mus l ired ) ak g l e b ly 1. er te e e , t Protocol - l d e x 1 27 n d e l . ) M r d e s l u i t i s g m . ) 5 l a m s s i n i e t o r e d 2 ( e l d i s l e b p r o u f C e b a T H h t · l o g n i s e e c u o t s u m d e t s u n i t o t ) e s ( ) l m / U j d e o r n . s n m a p 0 o i s r u M m o i a - e i t r 1 ( t x i y l a e h t e t s y c m 0 2 ( u l o s u t a r n a o t t s e g i d E n o i t u n i r e f l o s e p m e t e t o r e r s e m u l o v p n o i t u l o s . p e t s e m y z n e 2 k c o s e h t e t e l p m o c t s G A P - S D S . 1 5 o e h t i h t t e h T h t i w k c o t s e s a e l p n i r o f g n e x i m , d e g a p ) l m / r e v i l e d i r b U e r d e m y z n E l 1 ( n o i t c a e r i u q e r u o h s n i m a e t s y e m y z n e s c i l C H · e n i m a e t . e r u t a r e p m e t e h t n o i t u l o , x i d n e p p A . ) C ° e s a P A D e h t s y b t a h t s y c e e e m y f o , n o i t a r a p e r p h t d e f o m o o r 7 3 – 4 g n i r u d z n e m i t t a e ( n i E m y z n n o i g n i d d a t a s e f o E n o i t m y l p v i t c a h t y b e r u t a t u l i d u l i d z n E . n o i t a r a p e r p C A M I s i n i m . 5 1 0 2 / 7 0 e s a P A m a s 0 e g n e 0 n o i r e p l r e f o D 5 e s a P A D a 1 / t s e n i f f u 1 / 1 f o m e t 1 m y z B e k a t e v i t c a m r o f , n r t r o f a g i d a e n E l a 5 e s a P A D e m u l o o i t 1 d e e e v b u s m y e h t . n o i k o o b d n a H r i s e s a 1 g n . r e t n i h a b u e r i r t t u l v o m e r e l a e d x i t r a e t a b u c e t a b u c r a p e r r a p e r t Z G A T P P a w M n I P A D f I i w u D f I n I o s c s c n i S u d e c o e m y Z G A T r P . 1 . 2 . 3 . 4 . 6 . 5 Protocol 28 co exp 1. c In p 2 T Pr o r d 6 Ta T 3 4 ea he a ou f ig ilo . . . xH D C E bl ble mo d ot est ct nt nzy rse er t AP ys TAGZym esalte e is- ion exp Inc a the Incu DA (2 Mix i Sta wi Du so st im ion nc oc tag te 7. ai 7 nal a me c 0 lu case thin s a se r ubate en u an r P li e Rec in n ge m m are tion. t ol bate p per b d sts ase ys r /re 1 ts. g il ime the at Enz in ine M be d and ot is. 1 omm s v ion e t be 2 ). mg age DAPase he 5 olume prote g a En ·H nt f Ki yme car dige e or . for m tw lin xper IM z Cl t te in tr in co w o P nt e in h yme een a ried e mper f ndat in. ill as AC-purif ie re stion r ( 5 ar ( desalted length respondi ng of 20 10 im DAP give b 0 pa ra ti ve m scali out is preparation. En een ions en .3 ature mM U/ml) in si by act a zy me t. sati an t se of c o ng designe at ) ivate d for If adding prote d verify d DA Pa se time (4 sfac En u des 5 room t p ag preparati –37°C volume zyme pre mg/ of in tory ired, ged that th d by parati DA is the t e to he ml. te F cystea e result ). op sol w p s or en mperatu sta take co enzy ro Pa se The as t v tim ution o on, ab proces blishe st op ndi e t e S 50 s found be in DAPa al le ta mine amo me sa in tio . b rt p di mU use Suit (1 mples ing ring a d ro re. 5 ns d sing gest mi un 0 p in prep se ce to µl i d ig es a foun n (5 t x oi n poi U/ml ) T b ssin t o be in di he io thi the he le of to f µl) du ara gest n nt DA s d a g suf pilot protein 1 ts th c ti on en s rin to p and on preparat ive t ti P e m ep wi fic ro io v ase zy TAGZy me g g g t di e DA n e te ie . th iv me mp purification His th d t i nt io e n Enzyme fo r ig concent Pase 1 e ns c 1 f era - es so om or ta m r vol 0 ea d ti –1 gge ix lu pr ot tu com Ti et o digestio n ple ume Handbo ok ct ti t digestio n n. 00 re- ure tr e o rat ion (m d r a t 1 n mi e p If t adj o io – i ta U) m le on di cysteamine·H Cl ei ns d f t 10 n m o f ns rge te U at e or ed ges us to us sired, r th (1–10 d least experime nt. in a µ ted be t t SDS in ig per l n e t pr ion 07/2015 digestio n ge b estio th used desir e ote pro 10 -PAGE a e mg i n us µl p ti in n te mg me the ) ilot for ed ed of i in n Protocol - r o t m r e A T 29 o F n o e h t o r s n m % 0 5 g n r o f f o r h c N - o l y t c a u l o . x i r t a e v i t a t i t n i N e r e f e R c e h t m C A M I l l s n o a . d a g o n f . . i f e s n o i t a d n t e n n n f o o o i o c h r i i u q l b s e l g c a e e . n i e s e e v u r i r e f f u e m m a s f e r m t o t o t o m m u l u o B n n u o c d n r i u 1 r p r p l o p r p o v e a h e r g n s s n e t t e h a e e e m o v e i n i d t e o r i v i v i p t - w W o h c i p e t a r - t y Z d p u s o l f e h t t a n t a n n m u g r a t r e b w o l e v . s . p f e o f . n o i i t c a r t b u s e f o t G A T l o c h t e d n a d n t s l m g m i t a n a y a b l b a x 1 a t n o c - n i e n i m / l m d e s s e d e s s e e n o t i 5 b 1 e z i l i t n u e h t u s s u c n . 0 i g s n o i t a d n e t o r p i 5 . 0 r o . c o r p c o r p o t n i e v i t c a r e m h t ) h g n i k a h s n i n d e g g a t f o e h t i w e h t e h t u l o v e d t b u s ) s e m u l o v c e t o y b m m o c r e t e m a o t e m y z n i B - d e e r e f f u B i a t n o e t a r i d e r h g u o r h t b e a c . e 3 s i h h t n i a t e m y z n E ) e m u l o v t n m u s n i a t p g d e h t u l o n i p e n o w o l f o c n o c . ) t n o i s n e e n m u l o c n a n o c t s n m u l o c d e b a ® i c a m r a h l a d e e s a n i r u d m y z n E P s x 2 e m y Z G A d e l m n o s n o c s r e f e r 0 1 ( C L u ≥ p s u s T e r u t x i m i t c a e r m l m o r d e e f e s e P F n i e b m x 1 5 . 0 h s i n e s a P C A a e l P A D i t c a r f i t c a r f d l i f s ( n o i t c a r f c x e 5 p r o s e r n o i o t f o M I u o A D n s i s o r a g a h s r e m A ( f o h s i e e h t d l m t o n h g u h g u C L d e m r o f r e p A n t o h t i w t h t c a r f n o i t c a e r A T N e g a v a e l c 2 e g r p m u t c e - i e r u d o d l h g i e e b A T n a a f o , h N o c e c . s A T N - i N n n s t n m u o r h t - w o r h t - w t i w . n o i t l l o C g n i d n o p s o t k c g a t - s i e h 5 1 0 2 / 7 0 . t i k a c t a o l f o l f N - i N e r m u l o r p l o c l a v o m r o f H C L P F c a r p f d e e o c 2 h g u o r h t - e m e t s y s n m u n u o m a e h t e h 5 / e h r o c ( n o i t s e g i d t h t 1 5 e R , y h T h T . n i e t o t l o 5 , l a c d n n i w o l e l e r e g . e R r p f s e i l p p a r . * a y h p f o d n H ! t n ! t n h g u o v i t a r a c s i D 3 a l a e h n o i t a c i l p p a e m y Z G a a d e t g e h t r e n e g 1 t a r o e p A f o g a p e h t . 4 d i k o o b d n a H n o i s n e e s o r n I T 2 , l o l o c o s v s u . r g o t r e h n r e r h t - w t r o p t r o p r b i l i u 7 g n i s n i a t n p s / 5 n i d n s p e t r p e d d s q 1 5 1 t o r o l f E a g A u s m I a m o r p o c i b m I u f e h T t s t S t l A s a P s e R h s a W A s e c o t p e R H o s n e l b c o r p A g a p e m y Z G A T * r P i h T . 2 a T . 3 . 1 . 4 . 5 Cleavage of N-terminal His tags using DAPase, Qcyclase, and pGAPase Enzymes This section is for the combined DAPase–Qcyclase and pGAPase Enzyme digestion. For the digestion of proteins with a natural stop point using DAPase Enzyme alone see Protocols 1 and 2. The data in Table 8 can be used as a rough guide to the amounts of DAPase and Qcyclase Enzyme necessary to completely digest the N-terminal tag of a recombinant protein. However, substantial differences in activity may be encountered from protein to protein (see above). Therefore, the figures in Table 8 should be considered as a starting point for the pilot experiments only, and not as a general rule for any protein and reaction scale.

Table 8. Times generally required for DAPase–Qcyclase digestion at the given enzyme- to-target protein ratio

Amount of Amount of Amount of Reaction Required target protein DAPase Qcyclase temperature reaction time

4°C 5 h 1 mg 50 mU 3 U 20–25°C 60 min 50 µg 2.5 mU 150 mU 37°C 30 min

Buffer preparation From a 10x stock solution (see Appendix, page 44) prepare enough 1x TAGZyme Buffer (20 mM sodium phosphate, pH 7.0; 150 mM NaCl) for desalting and the TAGZyme digestion. Approximately 50 ml 1x TAGZyme Buffer is sufficient for a small-scale experiment. Note: Tags containing glutamate or aspartate residues can only be efficiently cleaved at pH 6.0–6.5. In this case, prepare the TAGZyme Buffer at lower pH (20 mM sodium phosphate, pH 6.0–6.5; 150 mM NaCl). Following cleavage, the pH of the reaction mixture must be increased to between pH 6.7 and 7.5 to enable efficient removal of His-tagged enzymes by IMAC. This can be accomplished by the addition of ~1/100 volume of 1M Tris·Cl. Desalting Desalt your His-tagged protein using gel filtration, ultrafiltration dialysis, hollow-fibre filtration, or the method best suited to your needs. It is recommended that proteins are exchanged into 1x TAGZyme Buffer. Ni-NTA matrices show negligible leaching of nickel ions into the elution buffer (10). However, when using IMAC resins that bind metal ions more weakly, addition of 5–10 mM EDTA to the eluted protein fraction prior to the desalting procedure is recommended to remove nickel ions from the 6xHis tag.

30 TAGZyme Handbook 07/2015 Protocol 4. DAPase–Qcyclase digestion (small-scale pilot experiment) The amount of DAPase and Qcyclase Enzyme and the length of incubation time required for complete digestion of the His tag depend on the target protein and its concentration. Generally, digestion is complete after treatment for 30 minutes at 37°C with 50 mU DAPase and 3 U Qcyclase Enzyme per mg protein but the optimal conditions for the processing of a tagged protein must be determined in pilot experiments. Table 9 gives guidelines for the conditions required for digestion of 50 µg target protein. Protein concentration should be at least 0.3 mg/ml.

Table 9. Recommendations for pilot DAPase–Qcyclase Enzyme digestion experiments

Enzyme/reagent For processing of 50 µg His-tagged target protein in 30 minutes Starting point Titration range DAPase (1 U/ml)* 2.5 mU (2.5 µl) 0.5–5 mU (0.5–5 µl)

Qcyclase (50 U/ml) 150 mU (3 µl) 30–300 mU (0.6–6 µl) P r o

Cysteamine·HCl (2 mM)* 5 µl 1–10 µl t o c o l * Dilute an aliquot of the DAPase Enzyme and cysteamine stock solutions according to steps 1 and 2 of protocol 4.

It is recommended that the progress of each digestion be analyzed by SDS-PAGE. Take 5 µl samples from each reaction every 10 minutes, add SDS sample buffer to inactivate the enzyme, and load samples onto an SDS gel including an undigested control. Alternatively, or in addition to varying the enzyme concentration, the incubation temperature can be varied.

TAGZyme Handbook 07/2015 31 Procedure 1. Prepare a 1/10 dilution of DAPase Enzyme stock solution (10 U/ml) using 1x TAGZyme Buffer. 2. Prepare a 1/10 dilution of cysteamine·HCl stock solution (20 mM) using distilled water. 3. Mix 1 volume DAPase Enzyme solution (1 U/ml) with 2 volumes cysteamine·HCl (2 mM). DAPase Enzyme is activated by cysteamine in this step. 4. Incubate for 5 min at room temperature. The enzyme mixture must be used within 15 min of preparation. During DAPase preparation, bring the protein solution to the desired incubation temperature (4–37°C). 5. Add 1.2 volumes of Qcyclase (50 U/ml) to the prepared DAPase. 6. Start the digestion by adding the enzyme mix to the temperature-adjusted protein solution. l 7. Incubate for a length of time that should deliver complete digestion (see Table 8, o c

o page 30). If desired, take samples during the reaction for SDS-PAGE analysis. t o r If removal of DAPase and Qcyclase Enzymes is required, please refer to the P protocol for small-scale subtractive IMAC in the Appendix, page 51.

32 TAGZyme Handbook 07/2015 Protocol 5. Preparative DAPase–Qcyclase digestion The TAGZyme Kit has been designed to enable processing and purification of at least 10 mg of desalted and IMAC-purified tagged protein. Suitable protein concentrations in digestion reactions are between 0.3 and 5 mg/ml. The amounts of DAPase and Qcyclase Enzyme to be used for digestion per mg of desalted protein are established in the pilot DAPase–Qcyclase digestion experiment. Table 10 lists the corresponding volumes to be used in a preparative digestion per mg of His-tagged protein.

Table 10. Recommendations for preparative DAPase–Qcyclase digestion

Enzyme/reagent For processing of 1 mg His-tagged target protein

Starting point Titration range DAPase (10 U/ml) 50 mU (5 µl) 10–100 mU (1–10 µl) Qcyclase (50 U/ml) 3 U (60 µl) 0.6–6 U (12–120 µl) Cysteamine·HCl (20 mM) 5 µl 1–10 µl

In most cases a linear scaling up of the conditions found to give complete digestion in the pilot experiment will give satisfactory results in a preparative digestion. If desired, a time course can be carried out to verify the optimal digestion conditions determined in the pilot experiments. P r o t o c

Procedure o l 1. Mix 1 volume DAPase solution (10 U/ml) with 1 volume cysteamine·HCl (20 mM). DAPase is activated by cysteamine in this step. 2. Incubate for 5 min at room temperature. The enzyme mixture must be used within 15 min of preparation. During DAPase preparation, bring the protein solution and an aliquot of 1x TAGZyme Buffer to the desired incubation temperature (4–37°C). 3. Add 12 volumes of Qcyclase (50 U/ml) to the prepared DAPase. 4. Start the digestion by adding the enzyme mix to the temperature-adjusted protein solution. 5. Incubate for a length of time that was found to be sufficient for complete digestion in the pilot experiment. If desired, take samples during the reaction for SDS-PAGE analysis.

TAGZyme Handbook 07/2015 33 Protocol 34 3 2 * T by Pr 4 5 T 1 hi s ab . . . . . A pag pr n ot oce le HR e p sub st bi Pa Im f Im m Ste E Aga Wa co pr Add pr Alt pr Re is Th su (s ra s ocol ro 17 51. qu sin ep ee at nd 5/ ote ote ov sp nta e p p sus ss er r c to co l p ea sh ogr il g o o t , 2 D r In s ion. ens in ide na p 4. ibrate the r r the ose* in. in. tra pag o ins pe AP or ta ta a d 1 gen the f g ap tive la ge Disc y d i nt nt H nd 6. o on a a dige rger ap pl ie s p f eral, R e ctiv low- in f ! ! h for nd col . se– rote 3 ly, 5 th y 51 The The the a th (c /5 5). the R a umn rd e system, orr stion Qcy cl ase 2 co e mou t through in. pack he emoval F e 6xH for col lu Ni-NTA PLC flo flo the ca k es mn nts it. to C wit I um w-th w-th remo n ponding Ni MAC reaction ol co is-tagged N hei o a be an lect h f -NTA do lu n i- pr g ro ro mn 2 N c f ht with val ot Enzym r le Ag pe LC ugh ugh not TA ml action the ein shou (Ame r av ag e rforme aros IMA of to of mixtu of resin plea or 5 exce fraction(s). l frac frac d DA 0.5 1 sham ml e be pyrogl u e C x DAPase se f FPLC rom step Pase tions tions r ed d TAGZy r s ≥ u (10 ref sca ea c m e us 2x s Pha du ed er l throu a p bed col i s le colu ti o s co ens rma to r and tep flow contai contai tamate i in fin um ng up th n lu me ci ion gh e this vol mn n ished mn 3 co n a Qc enz bed-vol reco di rate B Bi to um the n n ameter an u b volu ot subtractive yclase ta with ff y yme the the by ech) the mme e) er an in i of co s um d hak me into . o ng d processed processed lu 0.5 the is e p 0.5 r in Qcyclase n r mn t siz s s En u he ot uitabl in cubat dat ) 1 n TAGZy me one e ml/ ac 1 ei g zyme t and reco il a ml x mg i n-c step and ti on nd e the T on mi of io fo AG flo on be s. ve r ta s. c n n. t . f py py w-rate he su ol p lo of t d rg Z r t a ip ch o ed l W w- Handbo ok ro ro ym e in et di vo e ct e pG a gl gl i h t th ns sp n de recom Enzymes e vol u l p t en 1 ut ut u g h ro u AP ro osa B me ta e am am m re f uff u me rac u te in flow-t gg a gh mendatio l s bl q yl yl er. se in o o of e u t e f -e -e . i f no g an d ons 07/2015 matrix. the c x x Re fe r Enzyme N hro o a pr ten ten tita lo l i-NT u ns ote in c fro n 50% mns d d ugh hr tive g on F ed ed er or m to o A - Protocol 7. pGAPase digestion (small-scale pilot experiment) P

This step is only necessary for proteins that have been incubated with Qcyclase Enzyme. r o t o

Table 11. Times generally required for pGAPase reaction at the given enzyme-to-target c o protein ratio l

Target protein pGAPase Reaction temperature Required reaction time

4–8°C Overnight (15 h) 1 mg 1 U 20–25°C 3 h 50 µg 50 mU 37°C 90 min

Generally, digestion with pGAPase Enzyme is complete after treatment for 90 minutes at 37°C with 1.0–1.25 U of pGAPase Enzyme per mg of protein (see Table 11). However, optimal conditions may vary from protein to protein. Therefore, it is recommended that a titration of the enzyme concentration (see Table 12) or a control reaction with DAPase Enzyme (see “Analysis during the TAGZyme Procedure”, page 23) is carried out after completion of the TAGZyme procedure. Alternatively, vary time and/or temperature of incubation. Increase the incubation time when the incubation temperature is decreased.

Table 12. Recommendations for pilot pGAPase pyroglutamate removal experiments

Enzyme/reagent For processing of 50 µg pyroglutamyl-extended target protein

Starting point Titration range pGAPase (25 U/ml) 50 mU (2 µl) 10–100 mU (0.4–4 µl) Cysteamine·HCl (2 mM)* 2 µl 1–10 µl

* Dilute an aliquot of the cysteamine·HCl stock solution according to step 1 of protocol 7.

TAGZyme Handbook 07/2015 35 Protocol 36 5. 4. 3. 2. 1. i Ad p Incu o Mi x wa Pr s If Inc p n c f re GA a e r the p ubate e d te pa r p le yr bat moval 2 ar the Pa r . sub ogl flow µl e a e se tion. ac a c utamyl at tr f ys t Enz or 1/ 1 -thr act tivated of t he ea m iv pGAP 10 yme oug 0 e desired -e di lu in e IMAC min h xtended pGA is fraction ase ·H Cl ti on acti Pas at temperat i Enz n of vated (2 3 e the prot cy s 7°C. m Enzyme yme f rom M) Appendi te am in e· HC l ein. by u w The is the re. c it h ysteami re to For first quired, 2 mixture the x, µl time-period page subtractive of pyroglutamyl-e ne st pG AP as oc k ple in must 51 t ase so lu ti on his . step. be e I re TAGZy me guidel MAC. En z fe us r x (2 ym to ed te i 0 n nd es e t m h wi Handbo ok ed e ( M 2 re th 5 p ) fe pro ro us in U r to /m i ng t te 30 o co in l Tab ) d l pe contained min is ti l 07/2015 fo l r e r l 50 e s 11 af m d a . ter µ ll- g Protocol , e d a r t a . e f o e t r e t 37 g m s e n ( . n o i t f a s e n o a h t r e i t . 1 1 p n i a t n e i e v e w o e m m y z n E d e y m H o c t u n i m e l b e . a b u c n i l p m a ) l m / 0 0 o T d n e z n E f ) 1 s a l 3 9 U c n i e t g n i s u o c o 1 . d r t r 5 e s n m m o e y c e e s f e l t o r p a 2 ( o i h t r e f t Q r u f a i t a e b a c e a h e r r T w P A D n e m e m y t u o s i t i w r c e d t t a h t i e e s ( d z n E s i , d e e r t d e s u i w n r e p m e t d e d n e t x e - l a v o m e r r e r i r r a e b e c . p e t s s e n i l e d i u g n o i t e t f a e t a b u c s s u t a r i e t o r p i n i i h t e r o f e r e h T f o r o / d n a e s a P A G p c a e r t s u m n i ) 3 2 e f o e p m e t . n i e g m n e e b e r r l µ m i t u l y m a t u l g o r y p e t e l p m o c n o n o i t a b u c n i l o r t n o e p e g a p s i e t o r p c 0 4 t a m a t u l g e h t , r o e v a h e . C A M I n i m a e t x i m e F a o t y r a v o t h t i o r i t a b u c t s y c w r o t a h t n i , y l n i e e h T s y p ” e r u d e m y z n E t o y b m y z n E e v i t c a r n o e h t r p ) M m d n e m y z n E t b u . e r u t a r e p c o r P . n i e t o r p m i t a r t s e . C ° 7 3 e v i e s a P s e v i t a n r e t n i e t o r p 0 2 ( o r m e f e h w t t a a P t l A s r i f r o f t a r e e t a v i t c a A G p e s a P A G p d e a m i n e c n o c s i y r a v t f o e h t A G p d e d n e t x n i m e m e e U e m y Z G A T 5 1 0 2 / 7 0 h t i w y a r i s e d e - l m o y 5 y z 0 1 l C H · e n i m a m y r a s s e c d e t a r f m y z m y z e p e r P . e r u d e c o e h t s n o i 2 . e h t v i e n m a t s e t n e n E r p n E g t s y r o f n o i t a b u c y t a c n t u l . 8 t c a i . n o i t 1 – 0 l n n i e s e a u l e h e n o i t i d l µ t o e e g i d a r . 1 e n r u d e m y h t k o o b d n a H 4 h e t a b o s i g o r y s a h t a p f o s e h l o c c p t e e t a b u c l Z G d d f s t i w p e n a t i s y l a P A G p n I A x i M o u c n I e r p A i n o s T i t o t o m i s C ° a e r P A G , y l l a r e n e a r t p a n A t i e h e m y Z G A T p G i h T r P 7 3 o t “ t c n I . 1 . 2 . 3 . 4 Protocol 38 We 3 * T IM Pr 5 4 T 2 1 T T p a p hi s ab AGZym he os pp yro g . . . . . A pag pr n ot oce t- le AC lica T rec HR p e p st Pa co Add f Wa qu ch Ste Aga Alt pr Re su Imp Imp Eq AGZyme ra ro luta s ocol ro 17 51. sin ep 5/ ov sp om nta rom tio sus uilibr a ss er ce c to co l p sh g e or or t , ntit 2 m r In s ion. ens ide na 4. ns. the the ss ose* pag o men ins pe pro or ta ta at a gen the f 1 at tive la Disc togr at d i nt! nt! H e nd DAPase 9. on iv dige rger ap pl ie s p f ce eral, R e a e d low- in c re pr . rote e ly, nd 5 olumn Th Th 51 t dure a th investigatin (c a sidue /5 he oce dure the bin R a rd ph orr stion co e e e mou through in. p emoval F 2 for colu lu Ni-NTA fl fl d y PLC the a k , es mn ow ow in nts ck it. s to C page Enz can w system, ponding Ni reaction g by ol co mn N ith hei o -throu -throu a a lect f of -NTA lu i i- yme s pr ght subj mn 2 N c be f g w fini LC ot 23 th r le Ag TA ml action the th ein shoul ith (Ame r e gh gh av ag e ect ). shed e aros performed do IMA tre of or 6xHis- to of mixtu res plea 5 effi fraction(s). frac frac d ing atment 0.5 1x sham ml FPLC be in not e C p se f ci an rom tion tion r an TAGZym r s ≥ u ency (10 GAPase tagged ref sca ea c m e us 2x s d Pha e ed er l throu aliq s s p xcee t bed col co s le he colu ti o ens a rma to tep c c durin o in nd ont ont um lu up th n f uot de ci mn ion gh e t this vol d mn n pGAPase he 3 e co n a ai ai bed-vol reco ta S di b Bi g o a to n n um DS the ameter p gg uf b f ot volu subtractive w ta the the GAPa y t en flow ech) the f E he ith mme e) er ed in i co s um an nzyme zyme hak me into pr pr . o ng fu lu is e p 0.5 n al r Enzyme oc oc ll n r at mn siz rate se s s) u ot y uitabl ys in dat 1 nt TAGZy me iv one e e e ei 1 pr g En is s s e in ml and il a x mg se se i n-c step oc and on nd e of zy t cu pro (s he T of d d fo AG be es flo on ee s. . me bat by r ta c 0 n n t . fl te w-rate he su ol se p at at d t .5 ow rg Zy a i - ip i ch ca l A n d Handbo ok on i i e in ve ve et di vo e ct n m subtractive i -t a m n ta i s t . aly sp n recom hr at e l vol u p t l/ p p u 1 re ly g h W ro osa me ro ro b i o e mi si se ve ad m f u ug me rac te in s t t he flow-t ff e e d mendatio l n bl y pr i i h er. o of during of n. n. n removal t e f f t . i no ote in o or ons 07/2015 matrix. the c us Re fe r N hro o ens longer furthe r l i i-NT u ng ns fro 50% mns to ugh ure the on F of or m to A a a Troubleshooting Guide

Comments and suggestions

End product not sufficiently pure a) Poor quality of tagged Refer to The QIA expressionist for guidelines and protein preparation limitations of Ni-NTA IMAC purification of His-tagged proteins. b) Inefficient digestion When working with proteins purified by IMAC using matrices other than Ni-NTA, metal ions may be leached from the matrix. These metal ions may have an inhibitory effect on digestion. Include EDTA in the desalting proce - dure prior to digestion. Check N-terminal amino acid sequence for inefficiently cleaved dipeptides (Table 2, page 17). Check for the presence of DAPase Enzyme stop points in odd-numbered positions in the protein sequence. Check whether pH of the reaction is correct. The pH must be low - ered when Glu or Asp residues are in the tag sequence to be cleaved. Check the pH of the 1x TAGZyme Buffer preparation. pH optimum for the TAGZyme procedure is pH 6.7. to 7.5. Check for the presence of denaturants. Even low concentrations will inactivate DAPase Enzyme. Be sure that the length of the tag to be cleaved does not exceed 30 amino acids. The optimal DAPase Enzyme-to-protein ratio may vary from protein to protein. Determine optimum conditions as described in pilot experiments. In some cases, a certain percentage of the tagged protein is not cleaved, e.g., due to steric hindrance. This residual His-tagged protein will be efficiently removed by subtractive IMAC. c) Too many amino acids DAPase stop point is not present at correct position. removed (DAPase digestion Check sequence for correct positioning of stop continues into the body of point (Table 1, page 9). the target protein) When making use of glutamine (Q) stop point, be sure to include an excess of Qcyclase Enzyme in the incubation reaction with DAPase Enzyme. Refer to Table 8, page 30 for suitable ratio of DAPase Enzyme to Qcyclase Enzyme.

TAGZyme Handbook 07/2015 39 Comments and suggestions d) Continued digestion during Inefficient first-round subtractive IMAC can lead to pGAPase step DAPase Enzyme being present during pGAPase incubation. Be sure to equilibrate the IMAC column as described in the protocol. Be sure not to exceed the recommended flow rate of 0.5 ml/min. Check pH of column load as binding of DAPase and Qcyclase Enzymes may be inefficient at a pH lower than 7.0. Inefficient subtractive IMAC – refer to The QIA expressionist for general guidelines for IMAC and its limitations. e) Pyroglutamyl-extended pGAPase activity was inhibited. Be sure that urea protein remains in the is not present in the pGAPase reaction. final preparation Titrate pGAPase concentration to find out optimal concentration for complete digestion. Check for complete digestion by performing a DAPase control reaction as described on page 23. f) pGAPase Enzyme present Subtractive IMAC was inefficient. Refer to The after subtractive IMAC QIA expressionist for general guidelines for IMAC and its limitations.

40 TAGZyme Handbook 07/2015 Appendix Enzyme specifications DAPase Enzyme Recombinant dipeptidyl peptidase I, polyhistidine-tagged (DPPI/dipeptidyl aminopeptidase I/DAPI/Cathepsin C; EC 3.4.14.1). Source Recombinant baculovirus expression vector system expressing the cloned DPPI gene from rat liver Supplied form Solution in 3 mM sodium phosphate; 150 mM NaCl; 2 mM cysteamine·HCl; 50% glycerol, pH 6.7–7.0 Purity >98% (determined by SDS-PAGE) Concentration 10 units/ml Storage –20°C Stability 9 months at –20°C; 1 week at 2–8°C Assay conditions DAPase Enzyme is assayed at 37°C in 20 mM citric acid, 150 mM NaCl, 1 mM EDTA, 5 mM cysteamine, pH 4.5, containing 4 mM Gly-Phe- p-nitroanilide as substrate. Unit definition One unit is defined as the amount of enzyme that converts 1 µmol of substrate per minute under the conditions used. Specific activity 7–11 unit/mg protein (protein determined by Bradford assay using BSA as standard)

TAGZyme Handbook 07/2015 41 Qcyclase Enzyme Recombinant glutamine cyclotransferase, polyhistidine-tagged (GCT; EC 2.3.2.5) Source Recombinant baculovirus expression vector system expressing the cloned glutamine cyclotransferase gene from papaya latex Supplied form Solution in 8 mM sodium phosphate; 150 mM NaCl; 50% glycerol, pH 7.0 Purity >98% (determined by SDS-PAGE) Concentration 50 units/ml Storage –20°C Stability 1 year at –20°C; 1 week at 2–8°C Assay conditions Qcyclase Enzyme (0.5–2 units) is assayed at 37°C in 50 mM Tris·HCl, pH 8.0, containing 2.5 mM Gln-Trp-Glu as substrate. The product (pyroGlu-Trp-Glu) is separated from the substrate by eluting the column with a linear NaCl gradient in 10 mM Tris·HCl, pH 8.0. The portion of Gln-Trp-Glu converted to pyroGlu-Trp-Glu detected at 280 nm is calculated from the respective peak areas. Unit definition One unit is defined as the amount of enzyme, which converts 1 µmol of substrate per minute under the conditions used.

Specific activity 70–110 units/mg protein (protein determined by A280 )

42 TAGZyme Handbook 07/2015 pGAPase Enzyme Recombinant pyroglutamyl aminopeptidase, polyhistidine-tagged (PGAP; EC 3.4.19.3) Source E. coli expressing the cloned pyroglutamyl aminopeptidase gene from Bacillus amyloliquefaciens Supplied form Solution in 6 mM Tris·HCl; 40 mM NaCl; 2 mM EDTA; 2 mM cysteamine·HCl; 50% glycerol, pH 8.0 Purity >98% (determined by SDS-PAGE) Concentration 25 units/ml Storage –20°C Stability 1 year at –20°C; 1 week at 2–8°C Assay conditions PGAP (0.01–0.025 Units) was assayed at 37°C in 20 mM Tris·HCl, 100 mM NaCl, 5 mM EDTA, 5 mM cysteamine, pH 8.0, containing 2 mM pyroglutamyl- p-nitroanilide as substrate. Unit definition One unit is defined as the amount of enzyme, which converts 1 µmol of substrate per minute under the conditions used.

Specific activity 4.5–7 units/mg protein (protein determined by A280 )

TAGZyme Handbook 07/2015 43 Solutions required

10x TAGZyme Buffer for 500 ml

200 mM NaH 2PO 4 13.80 g NaH 2PO 4·H 2O (MW 137.99 g/mol) 1.5 M NaCl 43.83 g NaCl (MW 58.44 g/mol) Adjust to desired pH with NaOH. Filter (0.2 µm) into a sterile storage vessel and store at 2–8°C. Note: Upon 1:10 dilution pH will shift from 6.3 to 7.0.

Use of blunt-end–cutting restriction enzymes to introduce various amino acids at the N-terminus of the mature protein Blunt-ended restriction enzyme sites can be introduced at the 5' end of the protein-coding sequence by PCR, and cloned into the TAGZyme pQE-1 vector. Different restriction enzyme sites can be utilized to incorporate specific amino acids at the N-terminus of the mature protein (see Table 13). After expression of the construct, the His tag and glutamine residue are removed using the TAGZyme System. For example, your (mature) protein of interest starts with the amino acid alanine (Ala). To introduce Ala into amino acid position 10 (immediately following the glutamine residue) in the TAGZyme pQE-1 vector, construct a primer containing the restriction enzyme sequence for Fsp I. A suitable sense PCR primer is shown below. GCA codes for the desired amino acid alanine, and the sequence TGC GCA forms the recognition sequence of Fsp I. Fsp I-restricted PCR products can be blunt-end ligated into Pvu II-restricted TAGZyme pQE-1 vector, to give a construct encoding for a glutamine residue in an odd-numbered position, followed by the desired N-terminal amino acid, in this case alanine.

PCR sense primer: Fsp l N-terminal amino acid sequence: 5' N NNN TGC GCA NNN NNN NNN NNN NNN 3' Al a1 aa 2 aa 3 aa 4 aa 5 aa 6

44 TAGZyme Handbook 07/2015 Table 13. Restriction enzyme recognition sequences for the incorporation of the indicated amino acid directly after the glutamine residue encoded by the TAGZyme pQE-1 vector or into the Pml I site in the MCS of the TAGZyme pQE-2 vector*.

Amino acid(s) to be Suitable restriction Restriction enzyme introduced enzyme recognition sequence

Any Mly I GAGTC (N) 5M NNN

CTCAG (N) 5L NNN Xaa

Any-Ser Xmn I GAAN NM NNTTC N CTTN NL NNAAG N Xaa-Ser

Any †-Cys Msl I CA Py N NM NN Pu TG N GT Pu N NL NN Py AC Any †-Cys

Any †-Trp CA Py N NM NN Pu TG N GT Pu N NL NN Py AC N Any †-Trp

Ala Fsp I TG CM GCA AC GL CGT Ala

Sfo I GG CM GCC CC GL CGG Ala

Afe I AG CM GCT TC GL CGA Ala

Arg Bsr BI GA GM CGG CT CL GCC Arg

Nru I TC GM CGA AG CL GCT Arg

Table continued overleaf

TAGZyme Handbook 07/2015 45 Table 13. Continued

Asn Hpa I GT TM AAC CA AL TTG Asn

Hin cII GT P yM Pu AC CA P uL Py TG Asn (AAC)

Gln-Pro ‡ MscI TG GM CCA AC CL GGT ‡Gln-Pro (CCA)

Stu I AG GM CCT TC CL GGA ‡Gln-Pro (CCT)

Gly Nae I GC CM GGC CG GL CCG Gly

Sma I CC CM GGG GG GL CCC Gly

Ile Eco RV GA TM ATC CT AL TAG Ile

Ssp I AA TM ATT TT AL TAA Ile

Leu Pvu II CA GM CTG GT CL GAC Leu

Lys Dra I TT TM AAA AA AL TTT Lys

Lys-Leu, Lys-Pro, Lys-His, Pme I GTT TM AAAC Lys-Gln, Lys-Arg CAA AL TTTG Lys-Leu/Pro/His/Gln/Arg

Lys-Phe, Lys-Leu, Lys-Ile, Swa I ATT TM AAAT Lys-Met,Lys-Val TAA AL TTTA Lys-Phe/Leu/Ile/Met/Val

Table continued overleaf

46 TAGZyme Handbook 07/2015 Table 13. Continued

Thr Sca I AG TM ACT TC AL TGA Thr

Tyr Bst Z17I GT AM TAC CA TL ATG Tyr

Val Bsa Al Py AC M GT Pu Py T GL CA Py Val

Btr I CA CM GTC GT GL CAG Val

Pml I CA CM GTG GT GL CAC Val

Sna BI TA CM GTA AT GL CAT Val

Serine in position 2: Psh AI GACN NM NNGTC N Ala-Ser, Arg-Ser, Gln-Ser, CTGN NL NNCAG N Glu-Ser, Gly-Ser, Leu-Ser, Xaa-Ser Lys-Ser, Met-Ser,Pro-Ser, Ser-Ser, Thr-Ser, Trp-Ser, Val-Ser

* This list may not be complete! Only 5- or 6-nucleotide cutters are listed. † Any amino acid except Asp, Asn, His, Tyr, Cys, and Phe can be inserted (refer to the foottnote below ‡ when Pro is incorporated). ‡ Use of this restriction site incorporates a proline residue in the position following the glutamine stop. Proline functions as a stop two positions ahead, thus generating a protein with the N-terminal amino acids Gln1- Pro2… Omit Qcyclase Enzyme from the DAPase reaction in this case, as Qcyclase Enzyme will convert the glutamine residue to pyroglutamate.

TAGZyme Handbook 07/2015 47 Recommended PCR cloning strategy for use of the vector pQE-TriSystem in combination with TAGZyme cleavage The pQE-TriSystem vector allows parallel expression of a His-tagged recombinant protein in E. coli , baculovirus-infected insect cells, and mammalian cells using a single construct. The vector encodes a C-terminal His tag (which cannot be removed by the TAGZyme System). To make use of this extremely versatile expression vector, an N-terminal His tag must be introduced by PCR. A cloning strategy and primer design recommendations are given below. • Clone into the most N-terminal restriction site, Nco I, to avoid the introduction of slowly cleaved dipeptides (see Table 2, page 17). • Design a 5' (sense) oligonucleotide primer including the Nco I-compatible 5' restriction site Bsp HI and including sequences coding for a 6xHis tag (the use of the Bsp HI site allows the insertion of a lysine residue as the next amino acid following the start methionine). • Include a C-terminal stop codon (UAA) after the last amino acid • Clone into any one of the C-terminal restriction sites (e. g., Hin dIII) Refer to The QIA expressionist (8) or visit us at www.qiagen.com for more details on the pQE-TriSystem vector.

Design of the 5' (sense) PCR primer with an introduced glutamine stop point* BspHl ↓ 5' XXX XTC ATG AAA CAC CAT CAC CAT CAC CAT CAG XXX XXX XXX XXX XXX XXX 3' MK HH HHHH Q aa1 aa2 aa3 aa4 aa5 aa6

* If the protein to be expressed contains a natural stop point within the amino acids aa1 – aa3 (see Table 1, page 9) this can be used as a DAPase Enzyme stop point instead of the glutamine residue.

Design of the 3' (antisense) PCR primer including a stop codon (TTA) for cloning into the Hin dIII site

Cloning into restriction sites located 3' of Nco I is not recommended, because this will result in the insertion of an alanine residue directly behind the initial methionine. Alanine does not efficiently prevent methionine processing (1) which may result in erroneously processed DAPase Enzyme digestion products. Furthermore, the second N-terminal dipeptide would be Ile-Ser which will be inefficiently cleaved (see Table 2, page 17).

Hi ndIII ↓ 5' XX XXA AGC TTA XXX XXX XXX XXX XXX XXX 3' aa n aa n– 1 aa n– 2 aa n– 3 aa n– 4 aa n– 5

48 TAGZyme Handbook 07/2015 Table 14. The genetic code

First Second position Third position position (5' end) UC A G(3' end) U Phe Ser Tyr Cys U Phe Ser Tyr Cys C Leu Ser Stop Stop A Leu Ser Stop Trp G

C Leu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G

A Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G

G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G

TAGZyme Handbook 07/2015 49 Table 15. E. coli codon usage table. For each codon, frequency of usage (%) and the corresponding amino acid are given

First Second position Third position UCAGposition

U 2.23 Phe 0.85 Ser 1.62 Tyr 0.52 Cys U 1.66 Phe 0.86 Ser 1.22 Tyr 0.65 Cys C 1.39 Leu 0.72 Ser 0.20 Stop 0.09 Stop A 1.36 Leu 0.89 Ser 0.02 Stop 1.52 Trp G

C 1.10 Leu 0.70 Pro 1.29 His 2.09 Arg U 1.11 Leu 0.55 Pro 0.97 His 2.20 Arg C 0.39 Leu 0.84 Pro 1.53 Gln 0.36 Arg A 5.26 Leu 2.32 Pro 2.88 Gln 0.54 Arg G

A 3.03 Ile 0.90 Thr 1.77 Asn 0.88 Ser U 2.51 Ile 2.34 Thr 2.17 Asn 1.61 Ser C 0.44 Ile 0.71 Thr 3.36 Lys 0.21 Arg A 2.53 Met 1.44 Thr 1.03 Lys 0.12 Arg G

G 1.83 Val 1.53 Ala 3.21 Asp 2.47 Gly U 1.53 Val 2.55 Ala 1.91 Asp 2.96 Gly C 1.09 Val 2.01 Ala 3.94 Glu 0.80 Gly A 2.59 Val 3.36 Ala 1.78 Glu 1.11 Gly G

His tags suitable for exoproteolytic cleavage by the TAGZyme System The following His tags have been shown to deliver excellent expression rates in E.coli and be well-suited for convenient cleavage using the TAGZyme System. For a more detailed overview, see reference 12.

Table 16. Amino acid and encoding nucleotide sequences for recommended His tags

1234567 89

Amino acid MKHH HHH H(Q) Codon ATG AAA CAT CAC CAT CAC CAT CAC (GCT) Amino acid MRHH HHH H(Q) Codon ATG CGT CAT CAC CAT CAC CAT CAC (GCT)

50 TAGZyme Handbook 07/2015 Small-scale subtractive IMAC • Pipet 50 µl Ni-NTA suspension (25 µl settled bed volume) into a microcentrifuge tube and equilibrate by spinning briefly (10 s), removing the supernatant, adding 500 µl TAGZyme buffer, spinning briefly (10 s), and removing the supernatant. • Add the TAGZyme Enzyme reaction mixture and incubate at 4°C or room temperature for 10 min on a shaker platform or an end-over-end shaker. Ensure that the suspension is mixed well. • To recover the target protein, spin briefly (10 s), and carefully remove the supernatant. Be sure not to disturb the Ni-NTA matrix, which contains the bound TAGZyme Enzyme.

Recommendations for subtractive IMAC scale-up The use of a chromatography system with which the flow rate can be precisely controlled is recommended. When processing larger columns the flow rate is a critical factor for the efficient binding of His-tagged proteins to the Ni-NTA matrix. In addition, the use of Ni-NTA Superflow is recommended as it can withstand higher back pressures (up to 10 bar = 1 MPa). Table 17 gives guidelines for flow rates and column dimensions to be used when scaling up subtractive IMAC.

Table 17. Recommended column dimensions and flow rates for Ni-NTA IMAC scale up

Column dimensions Amount of (internal Flow rate Linear flow rate protein to be Ni-NTA bed diameter x (ml/min) (cm/h) processed volume length) Load Wash Load Wash Up to 1 mg 0.5 ml 0.5 cm x 2.5 cm 0.5 1.0 150 300 50 mg 10 ml 1.6 cm x 5 cm 5 10 150 300 1 g 200 ml 5 cm x 10.2 cm 50 100 150 300

TAGZyme Handbook 07/2015 51 References 1. Dalbøge, H., Bayne, S., and Pedersen, J. (1990) In vivo processing of N-terminal methionine in E. coli . FEBS Lett. 266 , 1. 2. Bujard, H., Gentz, R., Lanzer, M., Stüber, D. Müller, M., Ibrahimi, I. Häuptle, M.T., and Dobberstein, B. (1987) A T5 promotor based transcription-translation system for the analysis of proteins in vivo and in vitro. Methods Enzymol. 155 , 416. 3. Stüber, D., Matile, H., and Garotta, G. (1990) System for high-level production in Escherichia coli and rapid purification of recombinant proteins: application to epitope mapping, preparation of antibodies, and structure-function analysis. In: Lefkovits, I. and Pernis, B., eds., Immunological Methods, vol. IV, New York: Academic Press, p 121. 4. Schwarz, E., Scherrer, G., Hobom, G., and Kössel, H. (1978) Nucleotide sequence of cro, cII and part of the 0 gene in phage lambda DNA. Nature 272 , 410. 5. Sutcliffe, J.G. (1979) Complete nucleotide sequence of the E. coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43 , 77. 6. Farabaugh, P.J. (1978) Sequence of the Iac I gene. Nature 274 , 765. 7. Gottesman, S., Halpern, E., and Trisler, P. (1981) Role of sulA and sulB in filamentation by Ion mutants of Escherichia coli K-12. J. Bacteriol. 148 , 265. 8. The QIA expressionist (03/2001). Handbook for high-level expression and purification of 6xHis-tagged proteins. QIAGEN, Hilden, Germany. 9. Sambrook, J. et al. (1989) Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory Press. New York. 10. Steinert, K., Artz, C., Fabis, R., and Ribbe, A. (1996) Comparison of chelating resins for purification of 6xHis-tagged proteins. QIAGEN News 1996 No. 5, 12. 11. Hirel, P.H.-H., Schmitter, J.-M., Dessen, P., Fayat, G., and Blanquet, S. (1989). Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. USA 86 , 8247. 12. Pedersen, J., Lauritzen, C., Madsen, M.T., and Dahl, S.W. (1999) Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases. Prot. Expr. Purif. 15 , 389.

52 TAGZyme Handbook 07/2015 Trademarks Patented or patent-pending technology and/or registered or registration-pending trademark of QIAGEN: QIAGEN ®, QIA express ®, QIA expressionist ™, Ni-NTA. Coomassie is a registered trademark of ICI Organics Inc. FPLC is a registered trademark of Amersham Pharmacia Biotech. TAGZyme, DAPase, pGAPase, and Qcyclase are trademarks of UNIZYME. Triton is a registered trademark of Rohm & Haas Inc. Tween is a registered trademark of ICI Americas Inc.

Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

Hoffmann-La Roche owns patents and patent applications pertaining to the application of Ni-NTA resin (Patent series: RAN 4100/63: USP 4.877.830, USP 5.047.513, EP 253 303 B1), and to 6xHis-coding vectors and His-labeled proteins (Patent series: USP 5.284.933, USP 5.130.663, EP 282 042 B1). All purification of recombinant proteins by Ni-NTA chromatography for commercial purposes, and the commercial use of proteins so purified, require a license from Hoffmann-La Roche.

TAGZyme technology is licensed under U.S. Patent No. 5,691,169, U.S. Patent No. 5,783,413 and E.U. Patent No. 00759931B1.

TAGZyme Handbook 07/2015 53 Ordering Information

Product Contents Cat. No.

TAGZyme Kit 0.5 Units DAPase Enzyme, 34300 30 Units Qcyclase Enzyme, 10 Units pGAPase Enzyme, 20 mM Cysteamine·HCl (1 ml), Ni-NTA Agarose (10 ml), 20 Disposable Columns

TAGZyme pQE Vector Set TAGZyme pQE-1 and -2 Vector DNA, 32932 25 µg each

TAGZyme 2.5 Units DAPase Enzyme, 34362 DAPase Enzyme (2.5 U) 20 mM Cysteamine·HCl (1 ml)

TAGZyme 50 Units DAPase Enzyme, 34366 DAPase Enzyme (50 U)* 20 mM Cysteamine·HCl (25 ml)

TAGZyme 150 Units Qcyclase Enzyme, 34342 Qcyclase/pGAPase 50 Units pGAPase Enzyme Enzymes (150 U/50 U)

TAGZyme 3000 Units Qcyclase Enzyme, 34346 Qcyclase/pGAPase 1000 Units pGAPase Enzyme Enzymes (3000 U/1000 U)*

Related products

Ni-NTA Agarose † 25 ml nickel-charged resin 30210 (25 ml) (max. pressure: 2.8 psi)

Ni-NTA Superflow † 25 ml nickel-charged resin 30410 (25 ml) (max. pressure: 140 psi)

Polypropylene Columns (1 ml) 50/pack, 1 ml capacity 34924

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

* Bulk enzyme quantities are customized products; delivery may take up to 6 weeks. Enzymes are also avail able in GMP-grade. Please inquire. † Available in bulk quantities; please inquire.

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