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(12) United States Patent (10) Patent No.: US 7,618,799 B2 Coleman Et Al

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(12) United States Patent (10) Patent No.: US 7,618,799 B2 Coleman Et Al US007618799B2 (12) United States Patent (10) Patent No.: US 7,618,799 B2 Coleman et al. (45) Date of Patent: Nov. 17, 2009 (54) BACTERIAL LEADER SEQUENCES FOR NCBI Report for Accession No.YP 346180, Direct Submission on INCREASED EXPRESSION Aug. 8, 2005. Huber, D., “Use of Thioredoxin as a Reporter to Identify a Subset of Escherichia coli Signal Sequences That Promote Signal Recognition (75) Inventors: Russell J. Coleman, San Diego, CA Particle-Dependent Translocation.” Journal of Bacteriology, 2005, (US); Diane Retallack, Poway, CA pp. 2983-2991, vol. 187 (9). (US); Jane C. Schneider, San Diego, Miot, M. and Betton, J., “Protein Quality Control in the Bacterial CA (US); Thomas M. Ramseier, Periplasm.” Microbial Cell Factories, 2004, pp. 1-13. Newton, MA (US); Charles D. Ma, Q., et al., “Protein Secretion Systems of Pseudomonas Hershberger, Poway, CA (US); Stacey aeruginosa and Pfluorescens.” Biochim. Biophys. Acta, Apr. 1, 2003, Lee, San Diego, CA (US): Sol M. pp. 223-233, vol. 1611, No. 1-2. Resnick, Encinitas, CA (US) Retallack, D.M., et al..."Transport of Heterologous Proteins to the Periplasmic Space of Pseudomonas fluorescens Using a Variety of (73) Assignee: Dow Global Technologies Inc, Midland, Native Signal Sequences, ’’ Biotechnol Lett, Oct. 2007, pp. 1483 MI (US) 1491, vol. 29, No. 10. Urban, A., et al., “DsbA and DsbC Affect Extracellular Enzyme Formation in Pseudomonas aeruginosa. J. Bacteriol. Jan. 2001, pp. (*) Notice: Subject to any disclaimer, the term of this 587-596, vol. 183, No. 2. patent is extended or adjusted under 35 Wang, H., et al., “High-level Expression of Human TFF3 in U.S.C. 154(b) by 0 days. Escherichia coli.” Peptides, Jul. 1, 2005, pp. 1213-1218, vol. 26, No. 7. (21) Appl. No.: 12/022,789 EMBL Database Accession No. AF057031, Submitted Apr. 2, 1998 (XP-002483318). (22) Filed: Jan. 30, 2008 NCBI Database Accession No. Q3KHI7. Direct Submission Aug. 2005 (SP-002483317). (65) Prior Publication Data * cited by examiner US 2008/O193974 A1 Aug. 14, 2008 Primary Examiner Maryam Monshipouri Related U.S. Application Data (74) Attorney, Agent, or Firm—Jarett Abramson; Alston & Bird LLP (60) Provisional application No. 60/887,476, filed on Jan. 31, 2007, provisional application No. 60/887,486, (57) ABSTRACT filed on Jan. 31, 2007. Compositions and methods for improving expression and/or (51) Int. Cl. secretion of protein or polypeptide of interestina host cell are CI2N 9/00 (2006.01) provided. Compositions comprising a coding sequence for a CI2N IS/00 (2006.01) bacterial Secretion signal peptide are provided. The coding CI2N L/04 (2006.01) sequences can be used in vector constructs or expression (52) U.S. Cl. ................. 435/183; 435/252.3:435/320.1; systems for transformation and expression of a protein or 536/23.1 polypeptide of interest in a host cell. The compositions of the (58) Field of Classification Search ................. 435/183, invention are useful for increasing accumulation of properly 435/252.3, 320.1; 536/23.2 processed proteins in the periplasmic space of a host cell, or See application file for complete search history. for increasing secretion of properly processed proteins from the host cell. In particular, isolated Secretion signal peptide (56) References Cited encoding nucleic acid molecules are provided. Additionally, U.S. PATENT DOCUMENTS amino acid sequences corresponding to the nucleic acid mol ecules are encompassed. In particular, the present invention 5,629, 172 A * 5/1997 Mascarenhas et al. ...... 435/69.7 provides for isolated nucleic acid molecules comprising 5,801,017 A * 9/1998 Werber et al. .............. 435/69.2 nucleotide sequences encoding the amino acid sequences FOREIGN PATENT DOCUMENTS shown in SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and the nucleotide sequences set forth in SEQID NO: 1. WO WO 2005/089093 A2 9, 2005 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, as well as variants and OTHER PUBLICATIONS fragments thereof. Paulson et al., Nature Biotechnology, 23, 873-878, 2005.* 68 Claims, 11 Drawing Sheets U.S. Patent Nov. 17, 2009 Sheet 1 of 11 US 7,618,799 B2 ??eIduu???00£MOCIduuou,dys-OqspMod U.S. Patent Nov. 17, 2009 Sheet 2 of 11 US 7,618,799 B2 U.S. Patent Nov. 17, 2009 Sheet 3 of 11 US 7,618,799 B2 U.S. Patent Nov. 17, 2009 Sheet 4 of 11 US 7,618,799 B2 U.S. Patent Nov. 17, 2009 Sheet 5 of 11 US 7,618,799 B2 U.S. Patent Nov. 17, 2009 Sheet 6 of 11 US 7,618,799 B2 zz00-89°d= U.S. Patent Nov. 17, 2009 Sheet 7 of 11 US 7,618,799 B2 |odK1pIJAA Á??Apevoud O O v IO/(n) AAIoe would U.S. Patent US 7,618,799 B2 U.S. Patent US 7,618,799 B2 8 U.S. Patent NOV, 17 9 2009 Sheet 10 of 11 US 7 9 618,799 B2 |S QZ?Š?I, |S 9 S 308 IS VYHN {S H |S 2, a U.S. Patent Nov. 17, 2009 Sheet 11 of 11 US 7,618,799 B2 US 7,618,799 B2 1. 2 BACTERIAL LEADER SEQUENCES FOR branes (see Agarraberes and Dice (2001) Biochim Biophys INCREASED EXPRESSION Acta. 1513:1-24; Muller et al. (2001) Prog Nucleic Acid Res Mol. Biol. 66:107-157). CROSS REFERENCE TO RELATED Strategies have been developed to excrete proteins from the APPLICATION cell into the supernatant. For example, U.S. Pat. No. 5,348, 867; U.S. Pat. No. 6,329, 172: PCT Publication No. WO This application claims the benefit of U.S. Provisional 96/17943; PCT Publication No. WO 02/40696; and U.S. Application Ser. Nos. 60/887,476, filed Jan. 31, 2007 and Application Publication 2003/0013150. Other strategies for 607887,486, filed Jan. 31, 2007, the contents of which are increased expression are directed to targeting the protein to herein incorporated by reference in their entirety. 10 the periplasm. Some investigations focus on non-Sec type secretion (see for e.g. PCT Publication No. WO 03/079007: REFERENCE TO SEQUENCE LISTING U.S. Publication No. 2003/0180937; U.S. Publication No. SUBMITTED ELECTRONICALLY 2003/0064.435; and, PCT Publication No. WO 00/59537). However, the majority of research has focused on the secre The official copy of the sequence listing is Submitted elec 15 tion of exogenous proteins with a Sec-type secretion system. tronically via EFS-Web as an ASCII formatted sequence list A number of secretion signals have been described for use ing with a file named “339398 SequenceListing..txt, created in expressing recombinant polypeptides or proteins. See, for on Jan. 17, 2008, and having a size of 28,000 bytes and is filed example, U.S. Pat. No. 5,914,254; U.S. Pat. No. 4.963,495; concurrently with the specification. The sequence listing con European Patent No. 0177343; U.S. Pat. No. 5,082,783; PCT tained in this ASCII formatted document is part of the speci Publication No. WO 89/10971; U.S. Pat. No. 6,156,552; U.S. fication and is herein incorporated by reference in its entirety. 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. 5,595,898; 5,698, FIELD OF THE INVENTION 435; and 6,204,023; U.S. Pat. No. 6,258,560; PCT Publica tion Nos. WO 01/21662, WO 02/068660 and U.S. This invention is in the field of protein production, particu 25 Application Publication 2003/0044906: U.S. Pat. No. 5,641, larly to the use of targeting polypeptides for the production of 671; and European Patent No. EP 0121352. properly processed heterologous proteins. Strategies that rely on signal sequences for targeting pro teins out of the cytoplasm often produce improperly pro BACKGROUND OF THE INVENTION cessed protein. This is particularly true for amino-terminal 30 secretion signals such as those that lead to secretion through the Sec System. Proteins that are processed through this sys More than 150 recombinantly produced proteins and tem often either retain a portion of the secretion signal, polypeptides have been approved by the U.S. Food and Drug require a linking element which is often improperly cleaved, Administration (FDA) for use as biotechnology drugs and or are truncated at the terminus. vaccines, with another 370 in clinical trials. Unlike small 35 As is apparent from the above-described art, many strate molecule therapeutics that are produced through chemical gies have been developed to target proteins to the periplasm of synthesis, proteins and polypeptides are most efficiently pro a host cell. However, known strategies have not resulted in duced in living cells. However, current methods of production consistently high yield of properly processed, active recom of recombinant proteins in bacteria often produce improperly binant protein, which can be purified for therapeutic use. One folded, aggregated or inactive proteins, and many types of 40 major limitation in previous strategies has been the expres proteins require secondary modifications that are inefficiently sion of proteins with poor secretion signal sequences in inad achieved using known methods. equate cell systems. One primary problem with known methods lies in the As a result, there is still a need in the art for improved formation of inclusion bodies made of aggregated proteins in large-scale expression systems capable of secreting and prop the cytoplasm, which occur when an excess amount of protein 45 erly processing recombinant polypeptides to produce trans accumulates in the cell.
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