(19) TZZ __ _T (11) EP 2 615 172 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: (2006.01) (2006.01) 17.07.2013 Bulletin 2013/29 C12N 15/78 C12Q 1/68 (21) Application number: 12198545.1 (22) Date of filing: 25.04.2008 (84) Designated Contracting States: • Schneider, Jane C. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Diego, CA 92104 (US) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Hershberger, Charles Douglas RO SE SI SK TR Fremont, CA 94536 (US) (30) Priority: 27.04.2007 US 914361 P (74) Representative: Harrison Goddard Foote LLP 4th Floor (62) Document number(s) of the earlier application(s) in Merchant Exchange accordance with Art. 76 EPC: 17-19 Whitworth Street West 08746833.6 / 2 142 651 Manchester M1 5WG (GB) (71) Applicant: Pfenex Inc. Remarks: San Diego, CA 92121 (US) •This application was filed on 20-12-2012 as a divisional application to the application mentioned (72) Inventors: under INID code 62. • Ramseier, Thomas M. •Claims filed after the date of filing of the application/ Newton, MA 02558 (US) after the date of receipt of the divisional application • Coleman, Russell John (Rule 68(4) EPC). San Diego, CA 92101 (US) (54) Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins (57) The present invention provides an array for rap- erologous protein of interest in a periplasm compartment, idly identifying a host cell population capable of producing or may secrete the heterologous protein extracellularly heterologous protein with improved yield and/or quality. through an outer cell wall. The strain arrays are useful The array comprises one or more host cell populations for screening for improved expression of any protein of that have been genetically modified to increase the ex- interest, including therapeutic proteins, hormones, a pression of one or more target genes involved in protein growth factors, extracellular receptors or ligands, pro- production, decrease the expression of one or more tar- teases, kinases, blood proteins, chemokines, cytokines, get genes involved in protein degradation, or both. One antibodies and the like. or more of the strains in the array may express the het- EP 2 615 172 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 615 172 A1 Description FIELD OF THE INVENTION 5 [0001] This invention is in the field of protein production, particularly to identifying optimal host cells for large-scale production of properly processed heterologous proteins. BACKGROUND OF THE INVENTION 10 [0002] More than 150 recombinantly produced proteins and polypeptides have been approved by the U.S. Food and Drug Administration (FDA) for use as biotechnology drugs and vaccines, with another 370 in clinical trials. Unlike small molecule therapeutics that are produced through chemical synthesis, proteins and polypeptides are most efficiently produced in living cells. However, current methods of production of recombinant proteins in bacteria often produce improperly folded, aggregated or inactive proteins, and many types of proteins require secondary modifications that are 15 inefficiently achieved using known methods. [0003] Numerous attempts have been developed to increase production of properly folded proteins in recombinant systems. For example, investigators have changed fermentation conditions (Schein (1989)Technology, Bio/ 7: 1141-1149), varied promoter strength, or used overexpressed chaperone proteins (Hockney (1994) Trends Biotechnol. 12:456-463), which can help prevent the formation of inclusion bodies. 20 [0004] Strategies have been developed to excrete proteins from the cell into the supernatant. For example, U.S. Pat. No. 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. Application Publication 2003/0013150. Other strategies for increased expression are directed to targeting the protein to the periplasm. Some investigations focus on non- Sec type secretion (see for e.g. PCT Publication No. WO 03/079007; U.S. Publication No. 2003/0180937; U.S. Publication No. 2003/0064435; and, PCT Publication No. WO 00/59537). 25 However, the majority of research has focused on the secretion of exogenous proteins with a Sec- type secretion system. [0005] A number of secretion signals have been described for use in expressing recombinant polypeptides or proteins. See, for example, U. S. Pat. No. 5,914,254; U.S. Pat. No. 4,963,495; European Patent No. 0 177 343; U.S. Pat. No. 5,082,783; PCT Publication No. WO 89/10971; U.S. Pat. No. 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. 5,595,898; 5,698,435; and 6,204,023; U.S. Pat. No. 30 6,258,560; PCT Publication Nos. WO 01/21662, WO 02/068660 and U. S. Application Publication 2003/0044906; U. S. Pat. No. 5,641,671; and European Patent No. EP 0 121 352. [0006] Heterologous protein production often leads to the formation of insoluble or improperly folded proteins, which are difficult to recover and may be inactive. Furthermore, the presence of specific host cell proteases may degrade the protein of interest and thus reduce the final yield. There is no single factor that will improve the production of all heter- 35 ologous proteins. As a result, there is a need in the art for identifying improved large- scale expression systems capable of secretingand properly processingrecombinant polypeptides to producetransgenic proteinsin properlyprocessed form. SUMMARY OF THE INVENTION 40 [0007] The present invention provides compositions and methods for rapidly identifying a host cell population capable of producing at least one heterologous polypeptide according to a desired specification with improved yield and/or quality. The compositions comprise two or more host cell populations that have been genetically modified to increase the expression of one or more target genes involved in protein production, decrease the expression of one or more target genes involved in protein degradation, express a heterologous gene that affects the protein product, or a combination. 45 The ability to express a polypeptide of interest in a variety of modified host cells provides a rapid and efficient means for determining an optimal host cell for the polypeptide of interest. The desired specification will vary depending on the polypeptide of interest, but includes yield, quality, activity, and the like. [0008] It is recognized that the host cell populations may be modified to express many combinations of nucleic acid sequences that affect the expression levels of endogenous sequences and/or exogenous sequences that facilitate the 50 expression of the polypeptide of interest. In one embodiment, two or more of the host cell populations has been genetically modified to increase the expression of one or more target genes involved in one or more of the proper expression, processing, and/or translocation of a heterologous protein of interest. In another embodiment, the target gene is a protein folding modulator. In another embodiment, the protein folding modulator is selected from the list in Table 1. [0009] In another embodiment, one or more of the host cell populations has been genetically modified to decrease 55 the expression of one or more target genes involved in proteolytic degradation. In another embodiment, the target gene is a protease. In another embodiment, the protease is selected from the list in Table 2. [0010] In one embodiment, nucleotide sequences encoding the proteins of interest are operably linked to aP. fluo- rescens Sec system secretion signal as described herein. One or more of the strains in the array may express the 2 EP 2 615 172 A1 heterologous protein of interest in a periplasm compartment. In certain embodiments, one or more strains may also secrete the heterologous protein extracellularly through an outer cell wall. [0011] Host cells include eukaryotic cells, including yeast cells, insect cells, mammalian cells, plant cells, etc., and prokaryotic cells, including bacterial cells such as P. fluorescens, E. coli, and the like. 5 [0012] As indicated, the library of host cell populations can be rapidly screened to identify certain strain (s) with improved yieldand/or quality ofheterologously expressed protein. Thestrain arrays areuseful for screeningfor improved expression of any protein of interest, including therapeutic proteins, hormones, a growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like. 10 BRIEF DESCRIPTION OF THE FIGURES [0013] Figure 1A depicts plasmid pDOW1261-2 used for engineering genomic deletion inP. fluorescens. Figure 1B is a 15 schematic drawing of the constructions of a gene X deletion. Figure 2 is a Western blot analysis of soluble cells fractions prepared at 0 and 24 hours post- induction (10 and 124, respectively) in Δprc1, ΔdegP2, ΔLa2 and the grpEdnaKJ co-expression strains (Example 6). The top arrows point to thefully assembled monoclonal antibody in theco- expressed strainsbut notin thecontrol (DC440). r = recombinant; n-r= nonrecombinant. 20 DETAILED DESCRIPTION [0014] The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown, Indeed, these inventions may be embodied in many 25 different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. [0015] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. 30 [0016] Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the invention.