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(12) Patent Application Publication (10) Pub. No.: US 2010/0048864 A1 Coleman Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2010/0048864 A1 Coleman Et Al US 20100048864A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0048864 A1 Coleman et al. (43) Pub. Date: Feb. 25, 2010 (54) BACTERIAL LEADER SEQUENCES FOR Publication Classification INCREASED EXPRESSION (51) Int. Cl. (75) Inventors: Russell J. Coleman, San Diego, C07K I4/2 (2006.01) CA (US); Diane Retallack, Poway, CI2P 2L/06 (2006.01) CA (US); Charles D. Hershberger, CI2N I/2 (2006.01) Poway, CA (US); Stacey Lee, San CI2N 15/63 (2006.01) Diego, CA (US) C7H 2L/04 (2006.01) Correspondence Address: (52) U.S. Cl. ..................... 530/326; 435/69.1; 435/252.3: Alston & Bird LLP 435/252.33:435/320.1536/23.7 Dow Global Technologies, Inc. Bank Of America Plaza, 101 South Tryon Street, (57) ABSTRACT Suite 4000 Charlotte, NC 28280-4000 (US) Compositions and methods for improving expression and/or secretion of a polypeptide of interest in a host cell are pro (73) Assignee: Dow Global Technologies Inc., vided. Compositions including a coding sequence for a bac Midland, MI (US) terial Secretion signal peptide are provided. The compositions of the invention are useful for increasing accumulation of (21) Appl. No.: 12/604,061 properly processed proteins in the periplasmic space of a host cell, or for increasing secretion of properly processed pro (22) Filed: Oct. 22, 2009 teins. In particular, isolated secretion signal peptide-encoding nucleic acid molecules are provided. Additionally, amino Related U.S. Application Data acid sequences corresponding to the nucleic acid molecules are encompassed. The present invention provides for isolated (62) Division of application No. 12/022,789, filed on Jan. nucleic acid molecules including nucleotide sequences 30, 2008, now Pat. No. 7,618,799. encoding the amino acid sequences shown in SEQID NO:2. (60) Provisional application No. 60/887,476, filed on Jan. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and the nucleotide 31, 2007, provisional application No. 60/887,486, sequences set forth in SEQID NO:1, 3, 5, 7,9,11, 13, 15, 17, filed on Jan. 31, 2007. 19, 21, and 23, as well as variants and fragments thereof. Patent Application Publication Feb. 25, 2010 Sheet 1 of 11 US 2010/0048864 A1 PCR disbC-skp from pooW3001 template primers incorporate Spel-HindIII restriction sites sligate into expression vector poOW 1169 disbCskp disbC-skip SENSE PRM | ANTISENSE prM - X of PCR dsbC-skip 52ibp Spei Xbal Xhol 9110 bp Plac / A f repb-mob20 mobC f oriTnic pDOW2258 • Skp-DsbC expression a dual-lacOtac promoter opyrf selectable marker orTric repB-mob20 F.G. 1 Patent Application Publication Feb. 25, 2010 Sheet 2 of 11 US 2010/0048864 A1 New pooW2258 (SKP into DC454) Sample #2B-2 and 2B-4 S and adder SeeBlue 23-2 23-4 Pius 2 FG, 2 Patent Application Publication Feb. 25, 2010 Sheet 3 of 11 US 2010/0048864 A1 FIG 3A Patent Application Publication Feb. 25, 2010 Sheet 4 of 11 US 2010/0048864 A1 FG. 3B Patent Application Publication Feb. 25, 2010 Sheet 5 of 11 US 2010/0048864 A1 C.S.F. Sixfifession Hest strain Targe: iskA3e3&ser. perplasmic WTG454 F&S3 F-retiree broth - 3EE 33:S-st's 83632; C}one sease Siste 8sciuttle a free gift) g broth terminus g i:832 ES:3; i: 8.33 3.23 E: E E FG. 4 Patent Application Publication Feb. 25, 2010 Sheet 6 of 11 US 2010/0048864 A1 EP 468. 002.2 r 24 hours post induct ior F.G. 5 Patent Application Publication Feb. 25, 2010 Sheet 7 of 11 US 2010/0048864 A1 phoAactivity Cell nomalized to OD1 12OOOOO -H negative Control A20V mutant 5 1OOOOOO. T. r. v . S . 2 800000 600000 s CC 2 400000 O wild type 2OOOOO ---------- 0.02 -------- H ---H------- : O 1O 2O 3O 40 50 Time after induction (hrs) F.G. 6 Patent Application Publication Feb. 25, 2010 Sheet 8 of 11 US 2010/0048864 A1 pbp-proinsulin-phoA expresson in p.f. with 0.3m MIPTG (Western) SO: nSO BrO SO inso BrO 64. 50 36 piNS-008-3 plNS-008-5 FG. 7 Patent Application Publication Feb. 25, 2010 Sheet 9 of 11 US 2010/0048864 A1 cyto 484; 44.1 kDa native 484: 47.6 kDa SO nS CFB SO nS CFB EP484-003 (cyto) EP484-004 (native) FG. 8 Patent Application Publication Feb. 25, 2010 Sheet 10 of 11 US 2010/0048864 A1 Neg CupA2 Cup B2 CupC2 TolB none NikA 5550 Trg2C S S S. S S E S T S Neg Cup A2 Cup B2 CupC2 Tol B none NikA Neg Bce Figi 5550 Tig2O 8124 S S S S FG. 9 Patent Application Publication Feb. 25, 2010 Sheet 11 of 11 US 2010/0048864 A1 FIG. 10 US 2010/0048864 A1 Feb. 25, 2010 BACTERIAL LEADER SEQUENCES FOR system. The Sec system is responsible for export of proteins INCREASED EXPRESSION with the N-terminal signal polypeptides across the cytoplas mic membranes (see Agarraberes and Dice (2001) Biochim CROSS REFERENCE TO RELATED Biophys Acta. 1513:1-24; Muller et al. (2001) Prog Nucleic APPLICATION Acid Res Mol. Biol. 66:107-157). 0001. This application is a divisional of U.S. patent appli 0008 Strategies have been developed to excrete proteins cation Ser. No. 12/022,789, filed Jan. 30, 2008, which claims from the cell into the supernatant. For example, U.S. Pat. No. the benefit of U.S. Provisional Application Ser. Nos. 60/887, 5,348,867; U.S. Pat. No. 6,329,172: PCT Publication No. 476, filed Jan. 31, 2007 and 60/887,486, filed Jan. 31, 2007, WO 96/17943; PCT Publication No.WO 02/40696; and U.S. the contents of which are herein incorporated by reference in Application Publication 2003/0013150. Other strategies for their entirety. increased expression are directed to targeting the protein to the periplasm. Some investigations focus on non-Sec type REFERENCE TO SEQUENCE LISTING secretion (see for e.g. PCT Publication No. WO 03/079007: SUBMITTED ELECTRONICALLY U.S. Publication No. 2003/0180937; U.S. Publication No. 2003/0064.435; and, PCT Publication No. WO 00/59537). 0002 The official copy of the sequence listing is submitted However, the majority of research has focused on the secre electronically via EFS-Web as an ASCII formatted sequence tion of exogenous proteins with a Sec-type secretion system. listing with a file named “3801 11 SequenceListing..txt, cre 0009. A number of secretion signals have been described ated on Oct. 22, 2009, and having a size of 28,000 bytes and for use in expressing recombinant polypeptides or proteins. is filed concurrently with the specification. The sequence See, for example, U.S. Pat. No. 5,914,254; U.S. Pat. No. listing contained in this ASCII formatted document is part of 4.963,495; European Patent No. 0 177 343; U.S. Pat. No. the specification and is herein incorporated by reference in its 5,082,783; PCT Publication No. WO 89/10971; U.S. Pat. No. entirety. 6,156,552; U.S. Pat. Nos. 6,495,357; 6,509, 181; 6,524,827; 6,528,298; 6.558,939; 6,608,018; 6,617,143: U.S. Pat. Nos. FIELD OF THE INVENTION 5,595,898; 5,698,435; and 6,204,023; U.S. Pat. No. 6,258, 0003. This invention is in the field of protein production, 560; PCT Publication Nos. WO 01/21662, WO 02/068660 particularly to the use of targeting polypeptides for the pro and U.S. Application Publication 2003/0044906: U.S. Pat. duction of properly processed heterologous proteins. No. 5,641,671; and European Patent No. EP 0121352. 0010 Strategies that rely on signal sequences for targeting BACKGROUND OF THE INVENTION proteins out of the cytoplasm often produce improperly pro cessed protein. This is particularly true for amino-terminal 0004 More than 150 recombinantly produced proteins secretion signals such as those that lead to secretion through and polypeptides have been approved by the U.S. Food and the Sec System. Proteins that are processed through this sys Drug Administration (FDA) for use as biotechnology drugs tem often either retain a portion of the secretion signal, and vaccines, with another 370 in clinical trials. Unlike small require a linking element which is often improperly cleaved, molecule therapeutics that are produced through chemical or are truncated at the terminus. synthesis, proteins and polypeptides are most efficiently pro 0011. As is apparent from the above-described art, many duced in living cells. However, current methods of production strategies have been developed to target proteins to the peri of recombinant proteins in bacteria often produce improperly plasm of a host cell. However, known strategies have not folded, aggregated or inactive proteins, and many types of resulted in consistently high yield of properly processed, proteins require secondary modifications that are inefficiently active recombinant protein, which can be purified for thera achieved using known methods. peutic use. One major limitation in previous strategies has 0005 One primary problem with known methods lies in been the expression of proteins with poor secretion signal the formation of inclusion bodies made of aggregated pro sequences in inadequate cell systems. teins in the cytoplasm, which occur when an excess amount of 0012. As a result, there is still a need in the art for protein accumulates in the cell. Another problem in recom improved large-scale expression systems capable of secreting binant protein production is establishing the proper second and properly processing recombinant polypeptides to pro ary and tertiary conformation for the expressed proteins.
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