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(12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier Et Al US 200601 10747A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier et al. (43) Pub. Date: May 25, 2006 (54) PROCESS FOR IMPROVED PROTEIN (60) Provisional application No. 60/591489, filed on Jul. EXPRESSION BY STRAIN ENGINEERING 26, 2004. (75) Inventors: Thomas M. Ramseier, Poway, CA Publication Classification (US); Hongfan Jin, San Diego, CA (51) Int. Cl. (US); Charles H. Squires, Poway, CA CI2O I/68 (2006.01) (US) GOIN 33/53 (2006.01) CI2N 15/74 (2006.01) Correspondence Address: (52) U.S. Cl. ................................ 435/6: 435/7.1; 435/471 KING & SPALDING LLP 118O PEACHTREE STREET (57) ABSTRACT ATLANTA, GA 30309 (US) This invention is a process for improving the production levels of recombinant proteins or peptides or improving the (73) Assignee: Dow Global Technologies Inc., Midland, level of active recombinant proteins or peptides expressed in MI (US) host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant (21) Appl. No.: 11/189,375 protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recom (22) Filed: Jul. 26, 2005 binant protein expression. The process can improve protein production or can improve protein quality, for example, by Related U.S. Application Data increasing solubility of a recombinant protein. Patent Application Publication May 25, 2006 Sheet 1 of 15 US 2006/0110747 A1 Figure 1 09 010909070£020\,0 10°0 Patent Application Publication May 25, 2006 Sheet 2 of 15 US 2006/0110747 A1 Figure 2 Ester sers Custer || || || || || HH-I-H 1 H4 s a cisiers TT closers | | | | | | Ya S T RXFO 1961. HSV RXFO 1957: HSU RXFO3987:CbpA RXD05455: HtpG Patent Application Publication May 25, 2006 Sheet 3 of 15 US 2006/0110747 A1 Figure 3 RXFO5399: DnaK RXFO5406: DnaJ Patent Application Publication May 25, 2006 Sheet 4 of 15 US 2006/0110747 A1 Figure 4 plph GH vs. nitA Time Point Comparison plph GH vs. nitA Strain Comparison GH Tine Point Comparison Patent Application Publication May 25, 2006 Sheet 5 of 15 US 2006/0110747 A1 Figure 5 (~~~~aegae| Patent Application Publication May 25, 2006 Sheet 6 of 15 US 2006/0110747 A1 Figure 6 2 . 2 2 Patent Application Publication May 25, 2006 Sheet 7 of 15 US 2006/0110747 A1 Figure 7 100 -0-DC369 (wt, hCGH) --DC372 (hs|U, hCGH) -A-DC271 (wt, pbp:hCGH) -HDC373 (hstU, pbp:hCGH) Hours Patent Application Publication May 25, 2006 Sheet 8 of 15 US 2006/0110747 A1 Figure 8 L s S. r f ve s S. O s. N f f has s s E s () N - Patent Application Publication May 25, 2006 Sheet 9 of 15 US 2006/0110747 A1 Figure 9 e s?t Lif s N- V) L | - s t f ss te E s f S. N s - - f f O f f as N e ver b s O sos s w Ns s - -- s L ha s r s Patent Application Publication May 25, 2006 Sheet 10 of 15 US 2006/0110747 A1 Figure 10 iO 5 : 25 siss 23a iii.is 3: 8 s 8 B SR F 83 R O y Patent Application Publication May 25, 2006 Sheet 11 of 15 US 2006/0110747 A1 Figure 11 6000.00 8 5000.00 C 3 5 4000.00 -0-DC369 3000.00 --DC206 hCGH 2000.00 -A-hslU hCH is 1000.00 0.00 Hours 0 5 6 22 48 16 Patent Application Publication May 25, 2006 Sheet 12 of 15 US 2006/0110747 A1 Figure 12 e - C2 as 5is aE R v O is C O) O. E ww * O. "I Go C i .. Z s -nos Y s H . JJ O) 9. 3 is w N is s 9. 9 5 s 5 S2 e R 5 1 is al Patent Application Publication May 25, 2006 Sheet 13 of 15 US 2006/0110747 A1 Figure 13 A. Plasmid CrOSS-in k- Region to be deleted (1917 bp) SUV Genome ust N 11. SS-505 bp 634 bp -1lasmid Allelic exchange mutagenesis hsiuV deletion plasmid (Tet", pyrf") Cross-in: Tet pDOW2050 • Cross out: 5- FOA tolerant (A hsil JV ) ef pyrf B. Tet resistant; FOA Sensitive N - Genome plasmid Cross-out C. FOA tolerant Patent Application Publication May 25, 2006 Sheet 14 of 15 US 2006/0110747 A1 Figure 14 Cls U C s S. -0-HJ104 (1) O -o-HJ104 (2) c C -A-HJ117 (1) 2 w -o-HJ117 (2) d 4. 20 30 Hours after induction Patent Application Publication May 25, 2006 Sheet 15 of 15 US 2006/0110747 A1 Figure 15 C CO st to qs s S. - c | Na- • O C el 2 C CO q US 2006/01 10747 A1 May 25, 2006 PROCESS FOR IMPROVED PROTEIN with lac gene induction (Wei Y., et al. (2001) High-density EXPRESSION BY STRAIN ENGINEERING microarray-mediated gene expression profiling of Escheri chia coli. J Bacteriol. 183(2):545-56). Other groups have CROSS REFERENCE TO RELATED also investigated transcriptional profiles regulated after APPLICATION mutation of endogenous genes or deletion of regulatory genes (Sabina, J. etal (2003) Interfering with Different Steps 0001. This application claims priority to U.S. Provisional of Protein Synthesis Explored by Transcriptional Profiling Application No. 60/591489, filed Jul. 26, 2004. of Escherichia coli K-12J Bacteriol. 185:6158-6170; Lee J H (2003) Global analyses of transcriptomes and proteomes FIELD OF THE INVENTION of a parent strain and an L-threonine-overproducing mutant 0002 This invention is in the field of protein production, strain. J Bacteriol. 185(18):5442-51; Kabir M. M. et al. and in particular is a process for improving the production (2003) Gene expression patterns for metabolic pathway in levels of recombinant proteins or peptides or improving the pgi knockout Escherichia coli with and without phb genes level of active recombinant proteins or peptides expressed in based on RT-PCR J Biotechnol. 105(1-2): 11-31; Eymann host cells. C., et al. (2002) Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and BACKGROUND transcriptome analysis. J Bacteriol. 184(9): 2500-20). 0003 More than 155 recombinantly produced proteins 0007 Gill et al. disclose the use of microarray technology and peptides have been approved by the U.S. Food and Drug to identify changes in the expression of stress related genes Administration (FDA) for use as biotechnology drugs and in E. coli after expression of recombinant chloramphenicol vaccines, with another 370 in clinical trials. Unlike small acetyltransferase fusion proteins (Gill et al. (2001) Genomic molecule therapeutics that are produced through chemical Analysis of High-Cell-Density Recombinant Escherichia synthesis, proteins and peptides are most efficiently pro coli Fermentation and “Cell Conditioning for Improved duced in living cells. In many cases, the cell or organism has Recombinant Protein Yield Biotech. Bioengin. 72:85-95). been genetically modified to produce or increase the pro The stress gene transcription profile, comprising only 16% duction of the protein. of the total genome, at high cell density was used to evaluate “cell conditioning strategies to alter the levels of chaper 0004. When a cell is modified to produce large quantities ones, proteases, and other intracellular proteins prior to of a target protein, the cell is placed under stress and often recombinant protein overexpression. The strategies for reacts by inducing or Suppressing other proteins. The stress “conditioning involved pharmacological manipulation of that a host cell undergoes during production of recombinant proteins can increase expression of for example, specific the cells, including through dithiothreitol and ethanol treat proteins or cofactors to cause degradation of the overex mentS. pressed recombinant protein. The increased expression of 0008 Asai et al. described the use of microarray analysis compensatory proteins can be counterproductive to the goal to identify target genes activated by over-expression of of expressing high levels of active, full-length recombinant certain sigma factors that are typically induced after cell protein. Decreased expression or lack of adequate expres stresses (Asai K., et al. (2003) DNA microarray analysis of sion of other proteins can cause misfolding and aggregation Bacillus subtilis sigma factors of extracytoplasmic function of the recombinant protein. While it is known that a cell family. FEMS Microbiol. Lett. 220(1): 155-60). Cells over under stress will change its profile of protein expression, it expressing Sigma factors as well as reporter genes linked to is not known in any given example which specific proteins sigma factor promoters were used to show stress regulated will be upregulated or downregulated. gene induction. Microarrays 0009 Choi et al. described the analysis and up-regulation of metabolic genes that are down-regulated in high-density 0005 Microarray technology can be used to identify the batch cultures of E. coli expressing human insulin-like presence and level of expression of a large number of growth factor fusion protein (IGF-I) (Choi et al. (2003) polynucleotides in a single assay. See for eg. U.S. Pat. No. Enhanced Production of Insulin-Like Growth Factor I 6,040,138, filed Sep. 15, 1995, U.S. Pat. No. 6,344,316, filed Fusion Protein in Escherichia coli by Coexpression of the Jun. 25, 1997, U.S. Pat. No. 6,261,776, filed Apr. 15, 1999, Down-Regulated Genes Identified by Transcriptome Profil U.S. Pat. No. 6,403,957, filed Oct. 16, 2000, U.S. Pat. No. ing App. Envir. Microbio. 69:4737-4742). The focus of this 6,451,536, filed Sep. 27, 2000, U.S. Pat.
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