Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4367-4371, June 1989 Biochemistry Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II (human serum albumin binding/IgG binding) BJORN HAMMARBERG*, PER-AKE NYGREN*, ERIK HOLMGRENt, ANETTE ELMBLADt, MICHAEL TALLYf, ULF HELLMAN§, TOMAS MOKS*, AND MATHIAS UHLtN*¶ *Department of Biochemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden; tKabigen AB, S-112 87 Stockholm, Sweden; tDepartment of Endocrinology, Karolinska Institutet, S-104 01 Stockholm, Sweden; and §Ludwig Institute for Cancer Research, Biomedical Center, S-75 123 Uppsala, Sweden Communicated by Peter Reichard, February 28, 1989 (received for review September 27, 1988)

ABSTRACT A dual affinity fusion concept has been de- In this report, we describe the development of a concept in veloped in which the gene encoding the desired product is fused which the gene of interest is fused between two different between two flanking heterologous genes encoding IgG- and heterologous genes. After affinity purification of the dual albumin-binding domains. Using sequential IgG and serum affinity fusion protein by using both the N-terminal and the albumin affinity chromatography, a full-length tripartite fu- C-terminal domains, a full-length product is obtained, suit- sion protein is obtained. This approach was used to recover a able for structural and functional studies. The system can full-length fusion product in Escherichia coli containing the also be used to facilitate molecular studies of protein degra- human insulin-like growth factor II (IGF-ll). Surprisingly, the dation in vivo and in vitro, as both the C-terminal and the recombinant IGF-II showed increased stability against proteo- N-terminal regions can be recovered independently. lytic degradation in E. coli when produced as a dual affinity To develop an optimal dual affinity system, the two affinity fusion protein, as compared to an N-terminal fusion protein. tails should be small, soluble, and stable in various host After site-specific cleavage of the tripartite fusion protein, organisms and should preferably not form dimers or multi- IGF-ll molecules with immunological and receptor binding mers. It is advantageous for the tails to be secretion "com- activity were obtained without renaturation steps. The results petent" so that the product can be recovered from the culture demonstrate that proteins can fold into biologically active medium, which facilitates the protein purification and allows structures, even if provided with large flanking heterologous the formation of disulfide bonds. Finally, it is desirable that protein domains. The concept was further used to characterize the two affinity systems have similar binding strengths and do the degradation of recombinant IGF-II in this heter- not cross react. specific We have chosen a dual affinity fusion system based on the ologous host. IgG-binding domains of staphylococcal protein A and the albumin-binding domains of streptococcal protein G. This A large number of gene fusion systems to facilitate expres- system was used to express biologically active human insulin- sion and purification ofrecombinant proteins in heterologous like growth factor II (IGF-II) in Escherichia coli and also used hosts have been described (1). Most of these expression to characterize the specific degradation of recombinant IGF- systems have been designed for high expression levels to II in this heterologous host. yield insoluble material that accumulates as aggregates in the cytoplasm (1). The disadvantage of this "inclusion body" approach is that in vitro refolding is required to obtain a MATERIAL AND METHODS biologically active protein. Such refolding schemes are often Bacterial Strains and Vectors. E. coli HB101 (5) and RRI complex and demand specific and time-consuming optimiza- M15 (6) were used as hosts. Vectors used were phage tion for each gene product. M13mpl8 (Pharmacia) and plasmids pEMBL8, pEMBL9 (7), Alternative expression systems that yield a soluble gene pEZZ8 (8), pEZZT308 (9), pSPG2 (10), and pRIT18 (11). product with a native structure in vivo, therefore, have a great DNA Constructions. DNA work was carried out as de- advantage. In particular, protein engineering studies might scribed (5). The synthesis of oligonucleotides was performed benefit from an approach where a biologically active protein as described (12). DNA sequencing was performed as de- can be obtained directly. A soluble gene product also allows scribed (13). the assembly of fusion proteins containing an "affinity han- The plasmid pRIT24 was obtained by subcloning the al- dle" to facilitate the purification. The same general purifi- bumin-binding regions from streptococcal protein G derived cation scheme can thus be used for a variety ofgene products. from plasmid pSPG2. After digestion with EcoRI the ends For protein engineering studies this approach eliminates the were made blunt with the Klenow fragment of DNA poly- need for individual purification schemes for "mutant" pro- merase I, a synthetic Sal I linker (GGTCGACC; Pharmacia) teins even with altered biochemical properties (2). was added by ligation. Digestion with Sal I and Pst I yielded However, expression in heterologous hosts of soluble gene a 640-base-pair fragment that was isolated by agarose elec- products has been hampered by problems with proteolysis trophoresis and inserted between the same sites in pEMBL8. (3), in which a heterologous population of products is ob- The gene fragment encoding the albumin-binding domains tained even if an afflnity-purification approach is used (4). was isolated from this plasmid by digesting with EcoRI and The recovery of the full-length product from such a mixture HindIII, adjacent to the insertion sites. The expression requires additional purification steps and often gives low vector pEZZT308 was digested with EcoRI and HindIII and overall yield. This has emphasized the need for expression the fragment described above was inserted by ligation. The systems that ensure a full-length product. resulting plasmid, pRIT24, contains the staphylococcal pro-

The publication costs of this article were defrayed in part by page charge Abbreviations: HSA, human serum albumin; IGF, insulin-like payment. This article must therefore be hereby marked "advertisement" growth factor; RRA, radio receptor assay. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed.

4367 Downloaded by guest on September 29, 2021 4368 Biochemistry: Hammarberg et al. Proc. Natl. Acad. Sci. USA 86 (1989)

tein A promoter and signal sequence followed by a gene ...... encoding a dual affinity fusion protein consisting of the ZZ A ,,,,,, ,,,,.....B... region derived from staphylococcal protein A and the B1B2 B affinity purification O region of streptococcal protein G. The IGF-II gene was . 1 assembled from synthetic oligonucleotides and inserted into . ',Is the EcoRI and HindIII sites of pEZZ8 to yield plasmid pRIT19 (B.H., unpublished results). This plasmid was di- A affinity purification gested with Not I and Msp I and the 870-base-pair fragment Bound Flow through containing the noncoding region upstream of the promoter, the promoter region, and the regions encoding S, ZZ, and -n..... IGF-II was isolated. This fragment was ligated with a syn- Site-specific & cleavage thetic linker (5'-CGGCGAAATCTGAAATGG and its com- plementary sequence, 5'-GATCCCATTTCAGATTTCGC) A andE''''''ssssssssssssss...... B affinity Q purification to change the stop codon to an ATG triplet encoding a methionine residue, which enables CNBr cleavage (see Fig. 2B). The linker starts with the Msp I site in the IGF-II gene a new the B1B2 FIG. 1. Dual affinity fusion concept. The protein of interest (X) and ends with BamHI site in frame with with a putative protease degradation site (solid arrow) is fused region. The fragment was recovered by cleavage with EcoRI between two different affinity tails (A and B). and BamHI and the shorter fragment was purified and ligated with the large EcoRI-BamHI fragment isolated from radation ofrecombinant proteins can be obtained by selective pEMBL8. After transformation to E. coli RR1 M15, blue affinity purification. The flow-through fraction of the tail A colonies were isolated. DNA prepared from one of these affinity purification step yields the C-terminal degradation colonies was cut with Xho I and BamHI, cloned into products (Fig. 1), which can be N-terminally sequenced. In M13mp18, and sequenced. The EcoRI-BamHI fragment con- addition, "nicked" proteins that are proteolytically cleaved sisting ofthe mutated IGF-II gene without the stop codon was but held together by disulfide bonds can be released by isolated and ligated to the isolated fragment from pRIT24 to reduction of the material obtained by the dual affinity steps. yield the IGF-II dual affinity fusion plasmid, pRIT25. This might provide additional information about the specific Expression and Purification of Proteins. Bacteria were degradation sites. harvested by an osmotic shock procedure (14). The shock Dual Affinity Vector System. Nygren et al. (9) showed that lysate was passed through an affinity column of human serum the B1B2 fragment of the streptococcal protein G receptor albumin (HSA)-Sepharose (9) or of IgG-Sepharose Fast Flow binds specifically to HSA and also demonstrated that this (Pharmacia) as described (15). Flow-through and fractions fragment could be used to purify a fusion protein by HSA eluted with 0.5 M HOAc (pH 2.8) were collected and lyo- affinity chromatography. The similarities in size (-60 amino philized. Eluted material was lyophilized and dissolved in acid residues per binding unit), binding and elution condi- PBST buffer (50 mM sodium phosphate, pH 7.1/0.9% NaCl/ tions, stability, and solubility make the B1B2 domain and the 0.05% Tween 20) for a second affinity chromatography step ZZ domain of protein A suitable fusion partners in a dual or in 70% (vol/vol) formic acid for treatment with CNBr as affinity system. Both receptors are monomeric and show no described (4). cross affinity between the ligands (9). Protein Analysis. Total amount of protein was determined A suitable plasmid was constructed encoding the signal by spectrophotometric determination at 280 nm, using the peptide from protein A, ZZ (a synthetic IgG-binding fragment following extinction coefficients (cm2/mg): 0.37 for ZZ- derived from protein A), and B1B2 (an HSA-binding region IGF-II-B1B2, 0.17 for ZZ, 0.48 for IGF-II, and 0.45 for of protein G). The vector pRIT24 (Fig. 2A) contains unique B1B2. The immunological activity was determined by RIA cloning sites for EcoRI, Sma I, BamHI, and Sal I, which can (16) and IGF-II receptor binding activity by radio receptor be used to insert foreign genes between the two flanking gene assay (RRA) (17). Proteins were analyzed by NaDodSO4/ fragments encoding the IgG- and HSA-binding domains, PAGE on the Phast system (Pharmacia) and stained with respectively. The promoter and signal sequence of protein A Coomassie blue. Protein sequencing was performed as de- ensures efficient expression and secretion in several bacterial scribed by Guss et al. (10). hosts, including E. coli and Staphylococcus aureus (18). Mutagenesis of the Human IGF-H Gene. The gene for human IGF-II was chemically synthesized as a 240-base-pair RESULTS EcoRI-HindIII gene fragment, encoding the mature 67- Dual-Afminity Concept. An outline of the basic concept is amino acid residue IGF-II (A.E., unpublished results). The shown in Fig. 1. The gene encoding the protein ofinterest (X) synthetic gene was preceded by an ATG methionine codon is fused between two genes encoding two different affinity and terminated in a double TAA stop codon. Using two tails (A and B). In the example, the protein (X) has a synthetic oligonucleotides, the two stop codons were re- protease-sensitive site. A cell lysate containing the recom- placed by an ATG codon followed by a new BamHI restric- binant tripartite fusion protein is first passed through an tion site in the correct reading frame for the B1B2 domain affinity column containing a tail B-specific ligand. A mixture (Fig. 2A). The nucleotide and the deduced amino acid se- offull-length protein and proteolytic fragments containing the quences of the relevant parts of the mutated IGF-II gene are C-terminal fusion protein region can thus be obtained. In a shown in Fig. 2B. second passage through a tail A-specific affinity column, the The gene was transferred to the pRIT24 vector using the degraded proteins flow through while full-length fusion pro- unique EcoRI and BamHI restriction sites. The resulting tein is retained. After site-specific cleavage of the tails, the plasmid pRIT25 encodes a tripartite fusion protein with the protein of interest (X) is obtained by passing the cleavage structure schematically outlined in Fig. 2C. The two unique mixture through a mixed affinity column for tails A and B and methionine residues flanking the IGF-II gene product enable collecting the flow-through. site-specific cleavage with CNBr to release IGF-II. This The dual affinity fusion concept is attractive because it recombinant IGF-II has a native N terminus but contains a ensures recovery of full-length product and also facilitates cleavage artifact in the C terminus, either a homoserine or a characterization of gene products that are highly sensitive to homoserine lactone, depending on the pH of the buffer (19). host-specific proteases. Information about the specific deg- Also shown in Fig. 2C is the structure of the bipartite fusion Downloaded by guest on September 29, 2021 Biochemistry: Hammarberg et al. Proc. Natl. Acad. Sc. USA 86 (1989) 4369

A Sell A B A lEoRI -Mmil CO TOC CGA OGA TCC OTC GAC 1 2 3 4 5 Ala Asn Sir All Gly Sr Voi Asp 1 2 -9 3

- 6 _ /- 9 3 - 45 - 66 - 45 -31 - -31

_l~ Om- 21 -2 1 w- 1 4 1 4 FIG. 3. Reduced NaDodSO4/PAGE analysis of the bipartite (A) and the tripartite (B) fusion proteins affinity-purified from the peri- B Ecos gleEl Hindill plasmic space of E. coli. (A) Lanes: 1, IgG affinity-purified ZZ- G...... aaATMaT-3 IGF-II (pRIT19); 2, marker proteins with sizes in kDa. (B) Lanes: 1, total periplasmic proteins of E. coli containing pR25; 2, HSA ua vttzo Mt Gias affinity-purified ZZ-IGF-II-B1B2 fusion protein; 3, IgG affinity- linker by a synthetic purified fusion protein; 4, IgG and HSA affinity-purified fusion E*Rl * spi BsmHl protein; 5, flow-through of the HSA affinity column loaded with 5'.-...U...CT.. CPOOaaa.TCT:C~-TCC-3 material affinity-purified on IgG. The positions of marker proteins _ ...... OpysserGlu1lvsr are indicated with the sizes in kDa. observed after HSA chromatography (Fig. 3B, lane 2), C whereas a degradation product of 14 kDa corresponding to pRIT19 the ZZ domains was seen after IgG chromatography (Fig. 3B, lane 3). Thus the linker regions flanking the IGF-II moiety are susceptible to proteolysis. The material eluted from the IgG Z Meti3IGF-11 Met pRIT25 column (lane 3) was allowed to bind to an HSA column and the bound material and the flow-through were analyzed by FIG. 2. Schematic drawings ofthe dual affinity expression vector electrophoresis (Fig. 3B, lanes 4 and 5, respectively). As pRIT24 (A), mutagenesis of the IGF-ll gene (B), and the tripartite expected the low molecular mass bands from the IgG column protein encoded by pRIT25 compared with the N-terminal fusion did not bind to the HSA column (lane 5), and a highly purified protein encoded by pRIT19 with its methionine residues indicated full-length protein was obtained after the two sequential (C). Some relevant restriction sites are indicated. Boxes represent affinity steps (lane 4). the genes coding for the signal sequence (S), synthetic IgG-binding Site-Specific Cleavage ofthe Tripartite Protein. The region (Z), IGF-II, the HSA-binding region (Bi) and (B2), and affinity- P-lactamase (bla). The origin ofreplication ofE. coli is also indicated purified fusion protein was treated with CNBr to cleave at the (ori) as are promoters (arrows). In B mutagenized bases are marked two methionine residues flanking the IGF-II domain. The with stars. cleavage mixture was desalted, lyophilized, dissolved, and analyzed by IgG and/or HSA affinity chromatography and protein between the IgG-binding ZZ domains and the human NaDodSO4/PAGE (Fig. 4). The CNBr cleavage resulted in a IGF-11 gene (pRIT19). disappearance of the full-length 45-kDa band (lane 1) and Expression and Purification of the Bipartite and Tripartite yielded new bands of28 kDa and 14 kDa (lane 2) correspond- Fusion Proteins. E. coli cells containing either plasmid ing to the B1B2 and the ZZ domains, respectively. Only a pRIT19 or pRIT25 were grown overnight and the periplasmic weak band was observed corresponding to the size of the fraction was collected by osmotic shock. Analysis by IGF-II molecule (7.5 kDa), which suggests that a large NaDodSO4/PAGE (Fig. 3A, lane 1) shows that no or little fraction of the material was lost during the desalting and full-length protein was obtained for the ZZ-IGF-II bipartite Iyophilization steps. This might be due to the low solubility fusion protein, suggesting that human IGF-II is highly sus- of the recombinant IGF-II molecule without the flanking ceptible for proteases in E. coli. Surprisingly, analysis of the tripartite fusion protein (Fig. 3B, lane 1) demonstrates a 1 2 3 4 5 6 7 8 major band corresponding to the full-length fusion protein 93> ZZ-IGF-II-B1B2 (45 kDa), suggesting that the dual affinity 66> fusion concept stabilizes the recombinant protein against 45> _ proteolytic degradation. The lysate from the strain containing 31> the dual affinity fusion gene (Fig. 3B, lane 1) was divided into two parts and passed either through an HSA column or an 21> IgG column. Bound proteins were eluted and analyzed by 14> electrophoresis under reducing conditions (Fig. 3B, lanes 2 8> and 3). In both cases, the full-length protein was the major 6> product. Note that two bands of 31 and 23 kDa appeared when reduced material purified on both affinity columns was analyzed. These two bands were not observed by nonreduc- ing NaDodSO4/PAGE (data not shown) and thus suggest that FIG. 4. NaDodSO4/PAGE of cleaved tripartite fusion protein nicked full-length proteins were held together by disulfide under reducing conditions. Lanes: 1, ZZ-IGF-II-BIB2 before cleav- bonds. age; 2, after cleavage; 3, after cleavage flow-through of IgG column; 4, after cleavage eluate ofIgG column; 5, after cleavage flow-through The dual affinity approach allows comparison of degrada- of HSA column; 6, after cleavage eluate of HSA column; 7, after tion products purified by N-terminal or C-terminal affinity cleavage flow-through of mixed column; 8, after cleavage eluate of chromatography. A degradation product of 28 kDa corre- mixed column. The positions of marker proteins are indicated with sponding in size to the albumin-binding domain B1B2 was the sizes in kDa. Downloaded by guest on September 29, 2021 4370 Biochemistry: Hammarberg et al. Proc. Natl. Acad. Sci. USA 86 (1989)

heterologous domains. The results of various affinity purifi- A I cation steps with cleaved material are shown in lanes 3-8 (Fig. 4). The ZZ domains were retained by the IgG column (lane 4), and the B1B2 domains were retained by the HSA column (lane 6). The mixed affinity column retained both the ZZ and B1B2 parts (lane 8), and the IGF-II molecule was recovered as flow-through (lane 7). Nonreducing NaDod- S04/PAGE revealed that most material was full-length and monomeric, both before and after the site-specific cleavage (data not shown). Immunological and Receptor-Binding Activity. The correct N terminus of the IGF-II obtained after cleavage was con- firmed by N-terminal sequencing (data not shown). To show S- -4 -2 was a 0= that the recombinant product folded into biologically .0 active structure, despite the large heterologous flanking BC! 1092 dilution domains, the recombinant IGF-II (Fig. 4, lane 7) was ana- lyzed by a RIA using chicken anti-human IGF-II antisera and by a RRA using human placental membranes. Both assays I suggest a final recovery of active IGF-II molecules corre- 89 iIGF-II sponding to :10 gg of IGF-II per liter of culture medium. This value was low compared to the amount of produced tripartite fusion protein, confirming the analysis of the ma- terial after cleavage and desalting by the NaDodSO4/PAGE (Fig. 4, lane 2). The use of relatively hydrophobic solvents during the recovery of the cleaved material has improved the 48 - native IGF-1I final yield of biologically active IGF-II as measured by RRA to 10-20o (data not shown). The parallel curves in Fig. 5 A and B (RIA and RRA, respectively) show that native and 20 recombinant IGF-II compete with similar or identical affinity -3 -2 -1 9 with labeled native IGF-II. In addition, the specific RRA log1o dilution activity of the recombinant IGF-II obtained after a single purification by ion-exchange chromatography is approxi- FIG. 5. Analysis of IGF-II for immunological and receptor binding mately the same as purified native IGF-II obtained from activity using RIA (A) and RRA (B). Crude lyophilized recombinant human serum (data not shown). The results, therefore, dem- IGF-II was reconstituted in 50mM NH4OAc (pH 7.4) and added in 1:2 onstrate that the dual affinity fusion approach yields active (RIA) and 1:10 (RRA) dilution steps with material corresponding to 1 ,ug peptide hormone without any renaturation schemes. (A) and 10 ,ug (B) ofcleaved fusion protein as the initial sample (dilution Analysis of the Protease-Sensitive Site of IGF-ll in E. coli. step 0). Native IGF-II purified from human serum was used as standard Recombinant IGF-II has one or several protease-sensitive and the initial sample (dilution step 0) consisted of 10 ng (RIA) and 100 of native The standard devia- sites recognized by a host-specific protease in E. coli (B.H., ng (RRA) purified IGF-II, respectively. tions for various triplicate determinations are shown as bars. *, Re- unpublished results). The tripartite fusion protein obtained combinant IGF-II; O, native IGF-II. from E. coli can, therefore, be used to characterize the degradation products. Reduction of the disulfide bonds in the IGF-II in E. coli without a complex renaturation scheme. The affinity-purified fusion protein yields two smaller degradation results demonstrate that this peptide hormone, containing products (Fig. 3B, lane 4) that correspond to the two parts of a nicked tripartite molecule. The full-length fusion protein A purified by both IgG and HSA chromatography was, there- fore, reduced by 250 mM 2-mercaptoethanol to break all disulfide bonds. The reduced material was separated by size-exclusion chromatography under reducing conditions (Fig. 6A) into three major peaks corresponding to =45 kDa (I), 30 kDa (II), and =20 kDa (III). The protein material .0 CD from these peaks was recovered and the N-terminal se- 0 quences were determined by Edman degradation. The results 0 (Fig. 6A) revealed that peaks I and III contained the expected mBo N terminus of the tripartite protein, and peak II yielded a sequence starting with Arg-38 or Ser-39 of the native IGF-II. The proteolytic cleavage occurred in =75% of the product between the Arg-37 and the Arg-38, and 25% was cleaved between the Arg-38 and the Ser-39. Time The sequences and the sizes ofthe various peptides suggest a proteolytic site in the IGF-II moiety that yields degradation B products II and III (Fig. 6B). The fact that these degradation Z I Z IGF-11 | Bi I B2 1 products are obtained after reduction of a dual-affinity- purified material suggests that they correspond to the two parts of a nicked molecule held together by disulfide bridges. IIIIn

DISCUSSION FIG. 6. (A) Chromatogram of gel filtration of reduced full-length tripartite protein. The N-terminal sequences of corresponding peaks The present investigation shows that the dual affinity fusion are indicated using the single-letter amino acid code. (B) Peptides approach can be used to produce biologically active human resulting from proteolytic degradation. Downloaded by guest on September 29, 2021 Biochemistry: Hammarberg et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4371 three disulfide bonds, can fold into a biologically active the C-terminal region or both regions and can subsequently structure, even when flanked by relatively large affinity do- be characterized by protein sequencing and analysis. The mains in both the C- and N-terminal ends. The produced fusion protocol used in this paper, which involves purification ofthe protein has four parts, (i) a signal sequence from staphylo- full-length protein by two affinity steps followed by reduction coccal protein A, (ii) an IgG-binding region ofsynthetic origin, ofdisulfide bonds, affinity purification ofthe C-terminal end, (iii) the human IGF-II, and (iv) an albumin-binding region from and then N-terminal sequencing of the obtained fragment, streptococcal protein G. The fact that all of these parts of the can be used to gain information about the proteolysis site in fusion protein are functional demonstrates the remarkable any inserted protein. tolerance ofrecombinant fusion proteins to fold as long as the The dual affinity concept can also be used to answer individual domains of the peptides are intact. biological questions of more general nature. Experiments to Expression of recombinant IGF-II in E. coli using N- characterize error frequencies of ribosomes have been de- terminal fusion to the IgG-binding domain yielded a highly signed using this approach (L. Isaksson and M.U., unpub- degraded product (Fig. 3A, lane 1). In contrast, the dual lished results). The low solubility of recombinant human affinity approach yielded a mostly full-length tripartite fusion IGF-II (21) further accentuates the advantage of producing protein (Fig. 3B), and biological activity was obtained after heterologous proteins with soluble fusion partners. The fu- site-specific cleavage (Fig. 5). This interesting and surprising sion protein can be result suggests that the C-terminal affinity domain stabilizes recovered and purified from the cell lysate IGF-II. in a soluble form. In conclusion, the dual affinity fusion Native human IGF-II in vivo is produced with a C-terminal concept provides a general and simple tool to study transla- extension of 21 amino acid residues called region E (20). The tional and posttranslation modifications and facilitates the function of this region is still unknown, but it is tempting to expression of recombinant proteins with low solubility. speculate that its role is to stabilize the newly synthesized We are grateful to Drs. Bjorn Nilsson, Lennart Philipson, Maris IGF-II molecule from proteolysis or to facilitate folding in the Hartmanis, Staffan Josephson, and Lars Abrahmsdn for critical correct tertiary structure. The question arises whether the comments and advice. This investigation was supported by grants C-terminal affinity domain is able to contribute some of the from the Swedish Board ofTechnical Development, by the Swedish functions of the native region E. This may be accomplished Natural Research Council, and by the Swedish Medical Research by sterical protection, by protein interactions involving the Council (Grants 6897 and 4224). acidic protein G domain, or by influencing the secretion mechanism during the membrane translocation. 1. Marston, F. A. 0. (1986) Biochem. J. 240, 1-12. The analysis of the break-down products revealed that the 2. Carter, P. & Wells, J. A. (1987) Science 237, 394-399. protease responsible for the major degradation of human 3. Grodberg, J. & Dunn, J. J. (1988) J. Bacteriol. 170, 1245-1253. IGF-II in E. coli is probably an endoprotease with a speci- 4. Olsson, H., Lind, P., Pohl, G., Henrichson, C., Mutt, V., ficity for basic amino acids (Fig. 6). Interestingly, the N- Jornvall, H., Josephson, S., Uhldn, M. & Lake, M. (1988) terminal sequencing revealed that most of the material was Peptides 9, 301-307. the 5. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular nicked after two arginine residues at position 37 and 38 of Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold mature IGF-II, but some molecules were nicked between Spring Harbor, NY). these two arginines. The presumptive protease may, there- 6. Langley, K. E., Villarejo, M. R., Fowler, A. V., Zamenhof, fore, have a trypsin-like activity with a preference to cleave P. J. & Zabin, I. (1975) Proc. Natl. Acad. Sci. USA 72, 1254- after one or two basic residues. These results have prompted 1257. us to investigate if the outer membrane protease OmpT, with 7. Dente, L., Cesareni, G. & Cortese, R. (1983) Nucleic Acids a specificity for basic residues (3), might be responsible for Res. 11, 1645-1655. the degradation. The fusion protein ZZ-IGF-II encoded by 8. L6wenadler, B., Jansson, B., Paleus, S., Holmgren, E., Nils- pRIT19 (Fig. 2C) is highly susceptible to degradation in a son, B., Moks, T., Palm, G., Josephson, S., Philipson, L. & wild-type E. coli strain (Fig. 3A, lane 1). In contrast, >80% Uhldn, M. (1987) Gene 58, 87-97. of the material was found to be full-length when produced in 9. Nygren, P.-A., Eliasson, M., Palmcrantz, E., Abrahmsen, L. & an ompT mutant strain (B.H., unpublished results), suggest- Uhldn, M. (1988) J. Mol. Recognition 1, 69-74. that the E. 10. Guss, B., Eliasson, M., Olsson, A., Uhldn, M., Frej, A.-K., ing proteolytic instability ofrecombinant IGF-II in Jornvall, H., Flock, J.-I. & Lindberg, M. (1986) EMBO J. 5, coli is indeed caused by the OmpT protease. This demon- 1567-1575. strates the importance of molecular tools, such as the dual 11. Elmblad, A., Josephson, S. & Palm, G. (1982) Nucleic Acids affinity approach, to study and characterize protease degra- Res. 10, 3291-3301. dation of recombinant proteins. 12. Moks, T., Abrahmsen, L., Holmgren, E., Billich, M., Olson, It is noteworthy that human IGF-I can be efficiently A., Uhldn, M., Pohl, G., Sterky, C., Hultberg, H., Josephson, expressed in E. coli without severe proteolysis using the S., Holmgren, A., Jornvall, H. & Nilsson, B. (1987) Biochem- protein A system (15). A comparison of the primary amino istry 26, 5239-5244. acid sequences of the structurally similar IGF-I and -II 13. Olsson, A. & Uhlen, M. (1986) Gene 45, 175-181. 14. Nossal, N. G. & Heppel, L. A. (1966)J. Biol. Chem. 241, 3055- reveals that the two arginines recognized by a protease in E. 3062. coli are present on both molecules. The fact that IGF-I 15. Moks, T., Abrahmsdn, L., Osterlof, B., Josephson, S., Ostling, molecules are relatively insensitive to proteolysis, therefore, M., Enfors, S.-O., Persson, I., Nilsson, B. & Uhlen, M. (1987) suggests that these regions are structurally different or follow BiolTechnology 5, 379-382. different folding pathways that influence their protease sus- 16. Enberg, G. & Hall, K. (1984) Acta Endocrinol. (Copenhagen) ceptibility. 107, 164-170. There are a number of reasons to use this dual affinity 17. Takano, K., Hall, K., Ritz6n, M., Iselius, L. & Sivertsson, H. fusion concept. Many applications involving heterologous (1976) Acta Endocrinol. (Copenhagen) 82, 449-459. expression of recombinant proteins a defined and 18. Abrahmsen, L., Moks, T., Nilsson, B., Hellman, U. & Uhlen, require M. (1985) EMBO J. 4, 3901-3906. homogenous material for immunizations, diagnostics, or pro- 19. Gross, E. (1967) Methods Enzymol. 11, 238-255. tein characterization. Although the dual affinity approach 20. Zumstein, P. P., Luthi, C. & Humbel, R. E. (1985) Proc. Natl. also yields nicked proteins, the system ensures that only the Acad. Sci. USA 82, 3169-3172. full-length product is recovered. Another important applica- 21. Furman, T. C., Epp, J., Hsiung, H. M., Hoskins, J., Long, tion, already discussed, is the study of proteolysis both in G. L., Medelsohn, L. G., Schoner, B., Smith, D. P. & Smith, vivo or in vitro. The protein can be purified using the N- or M. C. (1987) BiolTechnology 5, 1047-1051. Downloaded by guest on September 29, 2021