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US 2008O193974A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0193974 A1 Coleman et al. (43) Pub. Date: Aug. 14, 2008 (54) BACTERIAL LEADER SEQUENCES FOR Publication Classification INCREASED EXPRESSION (51) Int. Cl. CI2P 2L/04 (2006.01) (75) Inventors: Russell J. Coleman, San Diego, CI2N IS/II (2006.01) CA (US); Diane Retallack, Poway, CI2N IS/00 (2006.01) CA (US); Jane C. Schneider, San CI2N L/20 (2006.01) Diego, CA (US); Thomas M. C07K I4/00 (2006.01) Ramseier, Newton, MA (US); CI2N 9/90 (2006.01) Charles D. Hershberger, Poway, (52) U.S. Cl. .................... 435/69.1:536/23.2:435/320.1; CA (US); Stacey Lee, San Diego, 435/252.3; 435/252.34; 435/252.33: 530/350; CA (US); Sol M. Resnick, 435/233 Encinitas, CA (US) (57) ABSTRACT Correspondence Address: Compositions and methods for improving expression and/or secretion of protein or polypeptide of interestina host cell are ALSTON & BRD LLP provided. Compositions comprising a coding sequence for a BANK OF AMERICA PLAZA, 101 SOUTH bacterial Secretion signal peptide are provided. The coding TRYON STREET, SUITE 4000 sequences can be used in vector constructs or expression CHARLOTTE, NC 28280-4000 (US) systems for transformation and expression of a protein or polypeptide of interest in a host cell. The compositions of the (73) Assignee: DOW GLOBAL invention are useful for increasing accumulation of properly TECHNOLOGIES, INC., processed proteins in the periplasmic space of a host cell, or Midland, MI (US) for increasing secretion of properly processed proteins from the host cell. In particular, isolated Secretion signal peptide (21) Appl. No.: 12/022,789 encoding nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the nucleic acid mol (22) Filed: Jan. 30, 2008 ecules are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences Related U.S. Application Data shown in SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and (60) Provisional application No. 60/887,476, filed on Jan. 24, and the nucleotide sequences set forth in SEQID NO: 1. 31, 2007, provisional application No. 60/887,486, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, as well as variants and filed on Jan. 31, 2007. fragments thereof. Title: BACTERIAL LEADER SEQUENCES FORENCREASED EXPRESSION PCR disbC-skip from pdOW3001 template • primers incorporateSpe-HindIII restriction sites ligate into expression vector pDOW1169 disb-skip dsbC-skip SNSERM MTSENSE PRM Spel * T 1 Xhat Pta s \ \ PCR disbC-skip 521.bp Y N res. * \-" Spe : Xia X8. pDOW2258 tac f 9110 bp pyrf k e repc ? repsi-mob28 riots oriTnic pDOW1169 Y 8627 bp pDOW2258 Skp-DsbC expression • dual-lacOtac promoter pyrf selectable marker oriTic rape-mob20 Patent Application Publication Aug. 14, 2008 Sheet 1 of 11 US 2008/O193974 A1 NOISSOETHAXACIGSVOETHONIHOHSGON@?mòASHACIVATTVIH@LOV8:º[1]L. 69IINNOCJd Patent Application Publication Aug. 14, 2008 Sheet 2 of 11 US 2008/O193974 A1 NOISSOETHAXECIGSVOETHONINJOJSRONGQOSHS(HACIVATTVIHALOV8:3I1IL Patent Application Publication Aug. 14, 2008 Sheet 3 of 11 US 2008/O193974 A1 NOISSOETH?X3CIGSVOETHONIHOHSAONGHIMÕ™ISHACIVATITVIHALOVHºn?L Patent Application Publication Aug. 14, 2008 Sheet 4 of 11 US 2008/0193974 A1 Patent Application Publication Aug. 14, 2008 Sheet 6 of 11 US 2008/O193974 A1 uol?onpu??sodsunouvzlo0 Patent Application Publication US 2008/O193974 A1 NOISSTHAXACIESV?THONIHOHSOEHONGITYÒGISHEIGIVETTVIHALOV8:êI?J. O(n) MA33e would Patent Application Publication Aug. 14, 2008 Sheet 8 of 11 US 2008/O193974 A1 NOISSOETH?X3CIGSVOETHONIHOHSOEHONGITÕHSHACIVATITVTHALOV8:??u!). Patent Application Publication Aug. 14, 2008 Sheet 9 of 11 US 2008/O193974 A1 Patent Application Publication Aug. 14, 2008 Sheet 10 of 11 US 2008/O193974 A1 Patent Application Publication Aug. 14, 2008 Sheet 11 of 11 US 2008/O193974 A1 NOISSOETH?XÃGIAISV?THONIHOHSRONGIQOPHSMadyanTVTÆLLOV@:3[]].?. US 2008/O 193974 A1 Aug. 14, 2008 BACTERIAL LEADER SEQUENCES FOR mic membranes (see Agarraberes and Dice (2001) Biochim INCREASED EXPRESSION Biophys Acta. 1513:1-24; Muller et al. (2001) Prog Nucleic Acid Res Mol. Biol. 66:107-157). CROSS REFERENCE TO RELATED 0008 Strategies have been developed to excrete proteins APPLICATION from the cell into the supernatant. For example, U.S. Pat. No. 0001. This application claims the benefit of U.S. Provi 5,348,867; U.S. Pat. No. 6,329,172: PCT Publication No. WO 96/17943; PCT Publication No.WO 02/40696; and U.S. sional Application Ser. Nos. 60/887,476, filed Jan. 31, 2007 Application Publication 2003/0013150. Other strategies for and 60/887,486, 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. 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 “339398 SequenceListing..txt, cre 0009. A number of secretion signals have been described ated on Jan. 17, 2008, 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. One duce transgenic proteins in properly processed form. barrier is that bacterial cytoplasm actively resists disulfide bonds formation, which often underlies proper protein fold SUMMARY OF THE INVENTION ing (Derman et al. (1993) Science 262:1744-7). As a result, many recombinant proteins, particularly those of eukaryotic 0013 The present invention provides improved composi origin, are improperly folded and inactive when produced in tions and processes for producing high levels of properly bacteria. processed protein or polypeptide of interest in a cell expres 0006 Numerous attempts have been developed to increase sion system. In particular, the invention provides novel amino production of properly folded proteins in recombinant sys acid and nucleotide sequences for secretion signals derived tems. For example, investigators have changed fermentation from a bacterial organism. In one embodiment, the Secretion conditions (Schein (1989) Bio/Technology, 7:1141-1149), signals of the invention include an isolated polypeptide with varied promoter strength, or used overexpressed chaperone a sequence that is, or is Substantially homologous to, a proteins (Hockney (1994) Trends Biotechnol. 12:456-463). Pseudomonas fluorescens (P. fluorescens) secretion polypep which can help prevent the formation of inclusion bodies. tide selected from a mutant phosphate binding protein (pbp). 0007 An alternative approach to increase the harvest of a protein disulfide isomerase A (dsbA), a protein disulfide properly folded proteins is to secrete the protein from the isomerase C (dsbC), a Cup A2, a CupB2, a CupC2, a NikA, a intracellular environment.
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