1111111111111111 IIIIII IIIII 11111 1111111111 1111111111 11111 lllll lllll 111111111111111 11111111
`US 20060110747Al
`
`c19) United States
`c12) Patent Application Publication
`Ramseier et al.
`
`c10) Pub. No.: US 2006/0110747 Al
`May 25, 2006
`(43) Pub. Date:
`
`(54) PROCESS FOR IMPROVED PROTEIN
`EXPRESSION BY STRAIN ENGINEERING
`
`(60) Provisional application No. 60/591,489, filed on Jul.
`26, 2004.
`
`(75)
`
`Inventors: Thomas M. Ramseier, Poway, CA
`(US); Hongfan Jin, San Diego, CA
`(US); Charles H. Squires, Poway, CA
`(US)
`
`Correspondence Address:
`KING & SPALDING LLP
`1180 PEACHTREE STREET
`ATLANTA, GA 30309 (US)
`
`(73) Assignee: Dow Global Technologies Inc., Midland,
`MI (US)
`
`(21) Appl. No.:
`
`11/189,375
`
`(22) Filed:
`
`Jul. 26, 2005
`
`Related U.S. Application Data
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12Q 1168
`(2006.01)
`GOIN 33/53
`(2006.01)
`C12N 15174
`(2006.01)
`(52) U.S. Cl. ................................ 435/6; 435/7.1; 435/471
`
`ABSTRACT
`(57)
`This invention is a process for improving the production
`levels of recombinant proteins or peptides or improving the
`level of active recombinant proteins or peptides expressed in
`host cells. The invention is a process of comparing two
`genetic profiles of a cell that expresses a recombinant
`protein and modifying the cell to change the expression of
`a gene product that is upregulated in response to the recom(cid:173)
`binant protein expression. The process can improve protein
`production or can improve protein quality, for example, by
`increasing solubility of a recombinant protein.
`
`

`

`Patent Application Publication May 25, 2006 Sheet 1 of 15
`
`US 2006/0110747 Al
`
`Figure 1
`
`0
`co
`
`0 ......
`
`0
`~
`
`C)
`IJ')
`
`0
`"Q"
`
`'in ...
`.a:: -C
`
`0
`
`i
`"5
`u
`
`0 -= ... c» c::
`
`~
`
`0
`C"')
`
`~
`F
`
`0
`N
`
`C)
`
`,....
`
`Q
`
`C)
`0
`0 ,....
`
`0
`
`0 .....
`
`0 ,....
`
`0
`
`WU!,l!,00
`
`0
`00
`
`N u
`
`......
`N u
`
`C t .....
`C 1 0
`C ' cs,
`C f
`
`~
`
`N u
`
`0
`
`N u
`
`

`

`Patent Application Publication May 25, 2006 Sheet 2 of 15
`
`US 2006/0110747 Al
`
`:
`
`_,,..r
`
`Figure 2
`
`Clmmlr :2
`
`ciui.mrl5
`
`V
`
`==
`
`"'----------•;.1 RXF03987:CbpA
`
`RXF01961: HslV
`RXF01957: HslU
`
`RXD05455: HtpG
`
`

`

`Patent Application Publication May 25, 2006 Sheet 3 of 15
`
`US 2006/0110747 Al
`
`Figure 3
`
`~ ,. -~ ..,
`.
`
`A
`-
`
`.
`-,.
`-
`
`A
`
`~
`
`Cllsbtr1
`
`..
`
`«:llllmr2
`
`,CIIJds3.
`
`Cludor4
`
`~ ( j
`
`_ /
`
`Cll&tse.
`
`I
`
`I
`
`V
`
`I
`
`- ~
`
`~
`
`.._ ~
`
`Clur:hir 1
`
`Clsbirs
`
`•Clod«8
`
`CliJclartD
`
`..
`
`Cllmr11
`
`Cb.far12
`
`I
`
`i ~ ~ -
`I ~ .
`,. --
`.
`
`..
`
`~
`
`-
`-.
`
`- -
`
`_.,,,.. I........_,
`
`i.,--
`
`-
`
`-~
`
`/ ✓ e,... .....
`# #
`
`Q
`
`{Ji
`Q
`
`e " {\ ..... / /
`
`RXF05399: DnaK
`~ RXF05406: DnaJ
`
`{\ ....
`
`#
`
`

`

`Patent Application Publication May 25, 2006 Sheet 4 of 15
`
`US 2006/0110747 Al
`
`Figure 4
`
`pbp::hGH vs. nitA
`Time Point Comparison
`
`pbp::hGH ,rs. nitA
`strain Comparison
`
`11-0
`&.7
`dn8J
`za
`
`hGH
`Time Point Comparison
`
`

`

`Patent Application Publication May 25, 2006 Sheet 5 of 15
`
`US 2006/0110747 Al
`
`Figure 5
`
`Cl
`Cl
`
`r) '_ ;~
`
`Cl
`Ii!
`N
`
`Cl i I r•
`
`Cl
`Cl
`
`iii .. N
`
`Cl
`Cl
`ID
`~
`N
`
`B .,
`t
`
`Cl
`
`

`

`Patent Application Publication May 25, 2006 Sheet 6 of 15
`
`US 2006/0110747 Al
`
`Figure 6
`
`♦
`t
`
`t
`t
`
`

`

`Patent Application Publication May 25, 2006 Sheet 7 of 15
`
`US 2006/0110747 Al
`
`Figure 7
`
`DC369 (wt, hG H)
`-
`---- DC372 (hsll.J-, hGH)
`-.-DC271 (wt, pbp::hGH)
`--- DC373 (hsll.J-, pbp::hGH)
`
`0
`0
`CD
`C
`0
`
`0.01
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`Hours
`
`

`

`Patent Application Publication May 25, 2006 Sheet 8 of 15
`
`US 2006/0110747 Al
`
`IFigmre 8
`
`LO
`
`~
`
`M
`
`N
`
`I • ..,,.,
`
`0)
`
`I l 1
`
`11
`
`1
`
`I
`
`'
`
`~
`
`"""'
`
`sl~
`'(/)
`I
`~1~
`~1~
`
`1
`I I
`till
`~
`~ - - - - - - - - - - - - - - - - - '
`
`I
`I a
`
`I=
`&.
`"it
`
`r,n
`
`Q
`
`00
`
`I
`
`tX)
`
`N
`
`

`

`Patent Application Publication May 25, 2006 Sheet 9 of 15
`
`US 2006/0110747 Al
`
`Figure 9
`
`tJ)
`
`r./J
`........
`
`r./J
`
`CJ)
`
`L-
`J:.
`Q
`Lf)
`
`i:
`
`~
`
`b ~
`.c
`□
`M
`
`:i
`
`~
`~
`
`tJ)
`
`... ~
`I
`.c
`~
`rn
`:s
`
`I ti)
`.... ~
`.c
`(:0
`I r.lJ
`
`:§
`
`-
`
`-~
`
`:iii
`
`(/'J
`
`r./J
`
`ti)
`
`if)
`
`L-
`J:
`"q'
`
`s:
`
`:i
`
`... ~
`:::
`
`.i:
`0
`
`!.la
`
`:5 !;I
`
`iii:
`
`Cf,!
`
`a)
`
`I'--
`w
`
`Lf)
`
`'ii:;f'
`
`('I')
`
`r-,1
`
`--
`
`0,
`
`a;}
`r-,.
`
`!;Cl
`
`Lf)
`
`"q'
`
`(I')
`
`N
`....
`
`m
`co
`r-...
`
`!;Cl
`u,
`
`"1J'
`rn
`
`N
`......
`
`& ~ ;g
`
`?'"
`
`OJ
`,:-,,
`l,O M
`
`fa
`N
`
`OJ
`'I:'""
`
`,q,
`'I:'""
`
`

`

`Patent Application Publication May 25, 2006 Sheet 10 of 15
`
`US 2006/0110747 Al
`
`Figure 10
`
`£L
`
`' f t
`
`:::::,_
`
`:::c
`(!)
`.s::.
`
`ft
`
`iO -~
`••
`:::c
`(!)
`.s::.
`! "i ~~
`- -o
`a,
`< 0 ~ 1 1 ) ••
`0
`0
`C">
`..
`..... ::c
`(.)
`.....
`C-, -,(!)
`
`t + ;.s::.
`
`fa
`
`§
`a
`'
`
`§
`
`8
`~ a
`
`0
`
`0 a
`a
`
`00900
`
`

`

`Patent Application Publication May 25, 2006 Sheet 11 of 15
`
`US 2006/0110747 Al
`
`Figure 11
`
`6000.00
`
`4000.00
`
`Cl) 5000.00
`(.)
`C:
`Cl)
`(.)
`tn
`...
`Cl)
`0
`::, 3000.00
`ii:
`Cl)
`> 2000.00
`.:;
`cu
`G)
`0:::
`
`1000.00
`
`-+-DC369
`---- DC206 _hGH
`-.-hslU hGH
`
`0.00
`
`0
`
`20
`
`40
`
`60
`
`Hours
`
`Time
`0
`4
`6
`24
`30
`48
`52
`
`hslu%
`5
`17
`22
`33
`24
`16
`15
`
`

`

`Patent Application Publication May 25, 2006 Sheet 12 of 15
`
`US 2006/0110747 Al
`
`Figure 12
`
`0
`.2>
`0
`u
`~ ·u
`Q)
`C.
`fl) • Q)
`C
`Q)
`C)
`Q)
`C
`0
`
`Q)
`
`~ e Q.
`
`-Cl) e>
`
`~
`Q)
`.c
`CG
`..J
`
`'ii -C
`
`Cl)
`E
`·c:: a.,
`>< w
`
`Q.
`
`0 ... -C:
`
`0 u
`
`,,,
`
`G)
`>.
`C
`'E
`
`G)
`C,
`
`C
`0 .:
`a.
`·c::
`CJ
`~ ! e g
`·.,tJ_i ii
`
`>
`t
`. . , a.,
`-: 0::
`. ·:
`
`'a;
`.a
`.!!
`
`ca -C:
`
`Cl)
`
`E
`·c::
`Cl)
`Q.
`><
`(I)
`C: ·-....
`(I)
`..c:
`.2>
`..c:
`
`Cl)
`(.)
`C:
`CG
`
`.c ...
`0
`en .c
`CG
`'ii
`:::::,
`C"
`Cl)
`
`0
`(.)
`
`0 ... -C:
`C: ·-...
`Cl)
`..c:
`C)
`:E
`
`,n
`
`CD
`·= c
`.a -
`:::i
`E ca o
`o :::i E
`0 g <(
`
`z ·C
`
`(.)
`Cl)
`
`(I)
`
`N =s
`'i:
`.a
`>.
`::c
`
`

`

`Patent Application Publication May 25, 2006 Sheet 13 of 15
`
`US 2006/0110747 Al
`
`Figure 13
`
`A. Plasmid cross-in
`
`,_ _ ___ Region to be deleted ____
`(1917 bp)
`
`_..
`
`----~~~::==============~=--Genome
`~
`~ / /
`
`hs/UV
`
`~
`
`--~~
`
`~--id ______ __,
`
`Allelic exchange mutagenesis
`• hs/UV deletion plasmid (Tet+, pyrF+)
`Cross-in : TetR
`• Cross out: 5- FOA tolerant
`
`l
`
`B. Tet resistant; FOA
`sensitive
`
`I
`I
`~~~00
`..£2£.
`hslUV
`tel
`-----1·m:1111~,~r.f:J\/'M/'./\i::iiiiiik-· ::::::==============:::i~,~----Genome
`I
`
`C. FOA tolerant I
`
`plasmid cross-out
`
`

`

`Patent Application Publication May 25, 2006 Sheet 14 of 15
`
`US 2006/0110747 Al
`
`Figure 14
`
`3500
`
`Cl) 3000
`u
`c:: 2500
`Cl) u
`Ill
`f 2000
`0
`::,
`c;:: 1500
`
`Cl)
`
`-~ - 1000
`
`IV
`a;
`a::
`
`500
`
`0
`
`0
`
`20
`10
`30
`Hours after induction
`
`40
`
`-+--- HJ 104 (1)
`-o- HJ104 (2)
`-.-HJ117(1)
`---o- HJ117 (2)
`
`

`

`Patent Application Publication May 25, 2006 Sheet 15 of 15
`
`US 2006/0110747 Al
`
`Figure 15
`
`V
`
`tn ~1-
`en
`~,Cl)
`a, I Cl)
`
`V
`
`Cl}
`
`a]CIJ
`
`.... ~,-
`
`tn
`
`V
`
`en
`
`V
`N
`
`Cl}
`
`a,ICI)
`
`V
`
`0
`
`en
`
`en
`
`i i
`
`~ -:>
`m m
`C
`C
`0
`0
`
`i 1
`-
`
`w
`I
`0.. C)
`..c
`Q
`
`w
`....
`C.
`C)
`I -,
`~ m
`C
`0
`
`~
`j
`
`::,
`i:,
`0
`~
`C)
`C ·-i:,
`0
`.... ::,
`LL
`....
`0
`.c
`~
`
`

`

`US 2006/0110747 Al
`
`May 25, 2006
`
`1
`
`PROCESS FOR IMPROVED PROTEIN
`EXPRESSION BY STRAIN ENGINEERING
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`[0001] This application claims priority to U.S. Provisional
`Application No. 60/591,489, filed Jul. 26, 2004.
`
`FIELD OF THE INVENTION
`
`[0002] This invention is in the field of protein production,
`and in particular is a process for improving the production
`levels of recombinant proteins or peptides or improving the
`level of active recombinant proteins or peptides expressed in
`host cells.
`
`BACKGROUND
`
`[0003] More than 155 recombinantly produced proteins
`and peptides 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 peptides are most efficiently pro(cid:173)
`duced in living cells. In many cases, the cell or organism has
`been genetically modified to produce or increase the pro(cid:173)
`duction of the protein.
`
`[0004] When a cell is modified to produce large quantities
`of a target protein, the cell is placed under stress and often
`reacts by inducing or suppressing other proteins. The stress
`that a host cell undergoes during production of recombinant
`proteins can increase expression of, for example, specific
`proteins or cofactors to cause degradation of the overex(cid:173)
`pressed recombinant protein. The increased expression of
`compensatory proteins can be counterproductive to the goal
`of expressing high levels of active, full-length recombinant
`protein. Decreased expression or lack of adequate expres(cid:173)
`sion of other proteins can cause misfolding and aggregation
`of the recombinant protein. While it is known that a cell
`under stress will change its profile of protein expression, it
`is not known in any given example which specific proteins
`will be upregulated or downregulated.
`
`Microarrays
`
`[0005] Microarray technology can be used to identify the
`presence and level of expression of a large number of
`polynucleotides in a single assay. See for eg. U.S. Pat. No.
`6,040,138, filed Sep. 15, 1995, U.S. Pat. No. 6,344,316, filed
`Jun. 25, 1997, U.S. Pat. No. 6,261,776, filed Apr. 15, 1999,
`U.S. Pat. No. 6,403,957, filed Oct. 16, 2000, U.S. Pat. No.
`6,451,536, filed Sep. 27, 2000, U.S. Pat. No. 6,532,462, filed
`Aug. 27, 2001, U.S. Pat. No. 6,551,784, filed May 9, 2001,
`U.S. Pat. No. 6,420,108, filed Feb. 9, 1998, U.S. Pat. No.
`6,410,229, filed Dec. 14, 1998, U.S. Pat. No. 6,576,424, filed
`Jan. 25, 2001, U.S. Pat. No. 6,687,692, filed Nov. 2, 2000,
`U.S. Pat. No. 6,600,031, filed Apr. 21, 1998, and U.S. Pat.
`No. 6,567,540, filed Apr. 16, 2001, all assigned to Affyme(cid:173)
`trix, Inc.
`
`[0006] U.S. Pat. No. 6,607,885 to E. I. duPont de Nemours
`and Co. describes methods to profile and identify gene
`expression changes after subjecting a bacterial cell to
`expression altering conditions by comparing a first and
`second microarray measurement. Wei et al. used a microar(cid:173)
`ray analysis to investigate gene expression profiles of E. coli
`
`with lac gene induction (Wei Y., et al. (2001) High-density
`microarray-mediated gene expression profiling of Escheri(cid:173)
`chia coli. J Bacterial. 183(2):545-56). Other groups have
`also investigated transcriptional profiles regulated after
`mutation of endogenous genes or deletion of regulatory
`genes (Sabina, J. et al (2003) Interfering with Different Steps
`of Protein Synthesis Explored by Transcriptional Profiling
`of Escherichia coli K-12 J Bacterial. 185:6158-6170; Lee J
`H (2003) Global analyses of transcriptomes and proteomes
`of a parent strain and an L-threonine-overproducing mutant
`strain. J Bacterial. 185(18):5442-51; Kabir M M, et al.
`(2003) Gene expression patterns for metabolic pathway in
`pgi knockout Escherichia coli with and without phb genes
`based on RT-PCR J Biotechnol. 105(1-2): 11-31; Eymann
`C., et al. (2002) Bacillus subtilis functional genomics: global
`characterization of the stringent response by proteome and
`transcriptome analysis. J Bacterial. 184(9):2500-20).
`
`[0007] Gill et al. disclose the use of microarray technology
`to identify changes in the expression of stress related genes
`in E. coli after expression of recombinant chloramphenicol
`acetyltransferase fusion proteins (Gill et al. (2001) Genomic
`Analysis of High-Cell-Density Recombinant Escherichia
`coli Fermentation and "Cell Conditioning" for Improved
`Recombinant Protein Yield Biotech. Bioengin. 72:85-95).
`The stress gene transcription profile, comprising only 16%
`of the total genome, at high cell density was used to evaluate
`"cell conditioning" strategies to alter the levels of chaper(cid:173)
`ones, proteases, and other intracellular proteins prior to
`recombinant protein overexpression. The strategies for
`"conditioning" involved pharmacological manipulation of
`the cells, including through dithiothreitol and ethanol treat(cid:173)
`ments.
`
`[0008] Asai et al. described the use ofmicroarray analysis
`to identify target genes activated by over-expression of
`certain sigma factors that are typically induced after cell
`stresses (Asai K., et al. (2003) DNA microarray analysis of
`Bacillus subtilis sigma factors of extracytoplasmic function
`family. FEMS Microbial. Lett. 220(1):155-60). Cells over(cid:173)
`expressing sigma factors as well as reporter genes linked to
`sigma factor promoters were used to show stress regulated
`gene induction.
`
`[0009] Choi et al. described the analysis and up-regulation
`of metabolic genes that are down-regulated in high-density
`batch cultures of E. coli expressing human insulin-like
`growth factor fusion protein (IGF-If) (Choi et al. (2003)
`Enhanced Production of Insulin-Like Growth Factor I
`Fusion Protein in Escherichia coli by Coexpression of the
`Down-Regulated Genes Identified by Transcriptome Profil(cid:173)
`ing App. Envir. Microbio. 69:4737-4742). The focus of this
`work was on the metabolic changes that occur during
`high-density conditions after protein induction. Genes that
`were down regulated after induction of recombinant protein
`production during high density growth conditions were
`identified and specific metabolic genes that had been down(cid:173)
`regulated were expressed in cells producing recombinant
`IGF-If. The work showed that increasing metabolic produc(cid:173)
`tion of certain nucleotide bases and amino acids could
`increase protein production and that growth rates could be
`modified by increasing expression of a down-regulated
`metabolic transporter molecule. These strategies were
`designed to alter the cellular environment to reduce meta(cid:173)
`bolic stresses associated with the protein production gener(cid:173)
`ally or with high density culture.
`
`

`

`US 2006/0110747 Al
`
`May 25, 2006
`
`2
`
`Protein Degradation
`
`[0010] Unwanted degradation ofrecombinant protein pre(cid:173)
`sents an obstacle to the efficient use of certain expression
`systems. The expression of exogenous proteins often
`induces stress responses in host cells, which can be, for
`example, natural defenses to a limited carbon source. All
`cells contain a large number of genes capable of producing
`degradative proteins. It is not possible to predict which
`proteases will be regulated by a given host in response to
`expression of a particular recombinant protein. For example,
`the bacteria P. jluorescens contains up to 200 proteases and
`protease related proteins.
`
`[0011]
`In the cytoplasm of E.coli, proteolysis is generally
`carried out by a group of proteases and cofactor molecules.
`Most early degradation steps are carried out by five ATP(cid:173)
`dependent Hsps: Lon/La FtsH/Hf!B, ClpAP, ClpXP, and
`ClpYQ/HslUV (Gottesman S (1996) Proteases and their
`targets in Escherichia coli. Annu. Rev. Genet. 30:465-506).
`Along with FtsH (an inner membrane-associated protease
`the active site of which faces the cytoplasm), ClpAP and
`ClpXP are responsible for the degradation of proteins modi(cid:173)
`fied at their carboxyl termini by addition of the non-polar
`destabilizing tail AANDENYALAA (Gottesman S, et al.
`(1998) The ClpXP and ClpAP proteases degrade proteins
`with carboxyl-terminal peptide tails added by the SsrA(cid:173)
`tagging system. Genes Dev. 12: 1338-1347; Herman C, et al.
`(1998) Degradation of carboxy-terminal-tagged cytoplasmic
`proteins by the Escherichia coli protease Hf!B (FtsH). Genes
`Dev. 12:1348-1355).
`
`[0012] Several approaches have been taken to avoid deg(cid:173)
`radation during recombinant protein production. One
`approach is to produce host strains bearing mutations in a
`protease gene. Baneyx and Georgiou, for example, utilized
`a protease-deficient strain to improve the yield of a protein
`A-~-lactamase fusion protein (Baneyx F, Georgiou G.
`(1991) Construction and characterization of Escherichia coli
`strains deficient in multiple secreted proteases: protease III
`degrades high-molecular-weight substrates in vivo. J Bac(cid:173)
`teriol l 73: 2696-2703). Park et al. used a similar mutational
`approach to improve recombinant protein activity 30%
`compared with the parent strain of E. coli (Park S. et al.
`(1999) Secretory production of recombinant protein by a
`high cell density culture of a protease negative mutant
`Escherichia coli strain. Biotechnol. Progr. 15: 164-167). U.S.
`Pat. Nos. 5,264,365 and 5,264,365 describe the construction
`of protease-deficient E. coli, particularly multiply protease
`deficient strains,
`to produce proteolytically sensitive
`polypeptides. PCT Publication No. WO 90/03438 describes
`the production of strains of E. coli that include protease
`deficient strains or strains including a protease inhibitor.
`Similarly, PCT Publication No. WO 02/48376 describes E.
`coli strains deficient in proteases DegP and Pre.
`
`Protein Folding
`
`[0013] Another major obstacle in the production of recom(cid:173)
`binant proteins in host cells is that the cell often is not
`adequately equipped to produce either soluble or active
`protein. While the primary structure of a protein is defined
`by its amino acid sequence, the secondary structure is
`defined by the presence of alpha helixes or beta sheets, and
`the ternary structure by covalent bonds between adjacent
`protein stretches, such as disulfide bonds. When expressing
`recombinant proteins, particularly in large-scale production,
`
`the secondary and tertiary structure of the protein itself is of
`critical importance. Any significant change in protein struc(cid:173)
`ture can yield a functionally inactive molecule, or a protein
`with significantly reduced biological activity. In many cases,
`a host cell expresses folding modulators (FMs) that are
`necessary for proper production of active recombinant pro(cid:173)
`tein. However, at the high levels of expression generally
`required to produce usable, economically satisfactory bio(cid:173)
`technology products, a cell often can not produce enough
`native folding modulator or modulators to process the
`recombinant protein.
`
`[0014]
`In certain expression systems, overproduction of
`exogenous proteins can be accompanied by their misfolding
`and segregation into insoluble aggregates. In bacterial cells
`these aggregates are known as inclusion bodies. In E. coli,
`the network of folding modulators/chaperones includes the
`Hsp70 family. The major Hsp70 chaperone, DnaK, effi(cid:173)
`ciently prevents protein aggregation and supports the refold(cid:173)
`ing of damaged proteins. The incorporation of heat shock
`proteins into protein aggregates can facilitate disaggrega(cid:173)
`tion. However, proteins processed to inclusion bodies can, in
`certain cases, be recovered through additional processing of
`the insoluble fraction. Proteins found in inclusion bodies
`typically have to be purified through multiple steps, includ(cid:173)
`ing denaturation and renaturation. Typical renaturation pro(cid:173)
`cesses for inclusion body targeted proteins involve attempts
`to dissolve the aggregate in concentrated denaturant and
`subsequent removal of the denaturant by dilution. Aggre(cid:173)
`gates are frequently formed again in this stage. The addi(cid:173)
`tional processing adds cost, there is no guarantee that the in
`vitro refolding will yield biologically active product, and the
`recovered proteins can include large amounts of fragment
`impurities.
`
`[0015] One approach to reduce protein aggregation is
`through fermentation engineering, most commonly by
`reducing the cultivation temperature (see Baneyx F (1999)
`In vivo folding of recombinant proteins in Escherichia coli.
`In Manual of Industrial Microbiology and Biotechnology,
`Ed. Davies et al. Washington, DC: American Society for
`Microbiology ed. 2:551-565 and references therein). The
`more recent realization that in vivo protein folding is
`assisted by molecular chaperones, which promote the proper
`isomerization and cellular targeting of other polypeptides by
`transiently interacting with folding intermediates, and by
`foldases, which accelerate rate-limiting steps along the fold(cid:173)
`ing pathway, has provided additional approaches combat the
`problem of inclusion body formation (see for e.g. Thomas J
`Get al. (1997). Molecular chaperones, folding catalysts and
`the recovery of active recombinant proteins from E. coli: to
`fold or to refold. Appl Biochem Biotechnol, 66:197-238).
`
`[0016]
`In certain cases, the overexpression of chaperones
`has been found to increase the soluble yields of aggregation(cid:173)
`prone proteins (see Baneyx, F. (1999) Recombinant Protein
`Expression in E. coli Curr. Opin. Biotech. 10:411-421 and
`references therein). The process does not appear to involve
`dissolution of preformed recombinant inclusion bodies but is
`related to improved folding of newly synthesized protein
`chains. For example, Nishihara et al. coexpressed groESL
`and dnaJK/grpE in the cytoplasm to improve the stability
`and accumulation of recombinant Cryj2 (an allergen of
`Japanese cedar pollen) (Nishihara K, Kanemori M, Kita(cid:173)
`gawa M, Yanagi H, Yura T. 1998. Chaperone coexpression
`plasmids: differential and synergistic roles of DnaK-DnaJ-
`
`

`

`US 2006/0110747 Al
`
`May 25, 2006
`
`3
`
`GrpE and GroEL-GroES in assisting folding of an allergen
`of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl.
`Environ. Microbial. 64:1694). Lee and Olins also coex(cid:173)
`pressed GroESL and DnaK and increased the accumulation
`of human procollagenase by tenfold (Lee S, Olins P. 1992.
`Effect of overproduction of heat shock chaperones GroESL
`and DnaK on human procollagenase production in Escheri(cid:173)
`chia coli. JBC 267:2849-2852). The beneficial effect asso(cid:173)
`ciated with an increase in the intracellular concentration of
`these chaperones appears highly dependent on the nature of
`the overproduced protein, and success is by no means
`guaranteed.
`
`[0017] A need exists for processes for development of host
`strains that show improved recombinant protein or peptide
`production, activity or solubility in order to reduce manu(cid:173)
`facturing costs and increase the yield of active products.
`
`[0018]
`It is therefore an object of the invention to provide
`processes for improving recombinant protein expression in
`a host.
`
`[0019]
`It is a further object of the invention to provide
`processes that increase expression levels in host cells
`expressing recombinant proteins or peptides.
`
`[0020]
`It is another object of the invention to provide
`processes to increase the levels of soluble protein made in
`recombinant expression systems.
`
`[0021]
`It is yet another object of the invention to provide
`processes to increase the levels of active protein made in
`recombinant expression systems.
`
`SUMMARY
`
`[0022] A process is provided for improving the expression
`of a recombinant protein or peptide comprising:
`
`[0023]
`i) expressing the recombinant protein or peptide in
`a host cell;
`
`[0024]
`ii) analyzing a genetic profile of the cell and
`identifying one or more endogenous gene products that are
`up-regulated upon expression or overexpression of the
`recombinant protein or peptide; and
`
`[0025]
`iii) changing expression of one or more identified
`endogenous gene products by genetically modifying the cell.
`
`[0026] The process can provide improved expression as
`measured by improved yields of protein, or can improve the
`recovery of active protein, for example by increasing solu(cid:173)
`bility of the expressed recombinant protein, or a related
`protein or peptide.
`
`[0027] Using this process, it can be determined which of
`the many cellular proteins are "chosen" by the cell to
`compensate for the expression of the foreign recombinant
`protein, and this information can lead to development of
`more effective protein expression systems. For example, it is
`known that, typically, a cell will selectively upregulate one
`or more proteases to degrade an overexpressed recombinant
`protein. However, it cannot be predicted in advance which
`protease(s) the cell will upregulate to compensate for the
`stress caused by any given recombinant protein. Analysis of
`the cell's genetic profile by microarray or equivalent tech(cid:173)
`nology can identify which proteases are upregulated in a
`given cell in response to exogenous protein production. This
`information is then used to genetically modify the cell to
`
`decrease the expression of these particular proteases, while
`sparing other proteins that are useful or even necessary for
`cell homeostasis.
`
`[0028] As another example, a cell may selectively upregu(cid:173)
`late one or more folding modulators or cofactors to increase
`the folding capability or solubility of the recombinant pro(cid:173)
`tein. Again, it cannot be predicted in advance which folding
`modulators or cofactors will be selected in a given system to
`assist in the processing of a specific recombinant protein.
`Analyzing the genetic profile by microarray or equivalent
`technology allows identification of the folding modulators or
`cofactors that have been upregulated. Based on this infor(cid:173)
`mation, the cell is genetically modified to increase the
`expression of the selected folding modulators or cofactors
`preferred by the cell for the given recombinant protein. This
`modification can increase the percent of active protein
`recovered, while minimizing the detrimental impact on cell
`homeostasis.
`
`[0029] Therefore, the yield and/or activity and/or solubil(cid:173)
`ity of the recombinant protein can be increased by modify(cid:173)
`ing the host organism via either increasing or decreasing the
`expression of a compensatory protein (i.e. a protein that is
`upregulated in response to given cell stress) in a manner that
`is selective and that leaves whole other beneficial mecha(cid:173)
`nisms of the cell.
`
`[0030] The process can be used iteratively until the
`expression of active recombinant protein is optimized. For
`example, using the process described above, the host cell or
`organism is genetically modified to upregulate, down regu(cid:173)
`late, knock-in or knock-out one or more identified compen(cid:173)
`satory proteins. The host cell or organism so modified can
`then be cultured to express the recombinant protein, or a
`related protein or peptide, and additional compensatory
`proteins identified via microarray or equivalent analysis.
`The modified host cell or organism is then again genetically
`modified to upregulate, down regulate, knock-in or knock(cid:173)
`out the additional selected compensatory proteins. This
`process can be iterated until a host cell or organism is
`obtained that exhibits maximum expression of active and/or
`soluble protein without undue weakening of the host organ(cid:173)
`ism or cell. These steps for example can be repeated for
`example, one, two, three, four, five, six, seven, eight, nine,
`or ten or more times.
`
`[0031]
`In another embodiment, the process further com(cid:173)
`prises: iv) expressing the recombinant protein or peptide in
`a genetically modified cell. In yet another embodiment, the
`process further comprises: v) analyzing a second genetic
`profile of the genetically modified cell expressing recombi(cid:173)
`nant protein or peptide and identifying one or more addi(cid:173)
`tional gene products that are differentially expressed in the
`modified cell expressing recombinant protein or peptide. In
`a further embodiment, the process additionally comprises:
`vi) changing the expression of one or more identified
`additional gene products to provide a double modified cell.
`Optionally, the recombinant protein or peptide, or a related
`protein or peptide, can be expressed in the double modified
`cell. The differentially regulated gene products identified in
`the modified cell can be up- or down-regulated when com(cid:173)
`pared to the host cell or when compared to the modified cell
`not expressing recombinant protein or peptide.
`
`[0032]
`In yet another embodiment, the process further
`comprises: iv) analyzing a second genetic profile of a
`
`

`

`US 2006/0110747 Al
`
`May 25, 2006
`
`4
`
`genetically modified cell expressing recombinant protein or
`peptide and identifying one or more additional gene prod(cid:173)
`ucts that are differentially expressed in the modified cell that
`is not expressing recombinant protein or peptide. In a further
`embodiment, the process additionally comprises: v) chang(cid:173)
`ing the expression of one or more additional identified gene
`products in the modified cell to provide a double modified
`cell. The differentially regulated gene products identified in
`the modified cell can be up- or down-regulated when com(cid:173)
`pared to the host cell or organism or when compared to the
`modified cell not expressing recombinant protein or peptide.
`
`[0033]
`In one specific embodiment, a process is provided
`for improving the expression of a recombinant protein or
`peptide comprising: i) expressing the recombinant protein or
`peptide in a host cell; ii) analyzing a genetic profile of the
`cell and identifying at least one protease that is up-regulated
`when the recombinant protein or peptide is expressed; and
`iii) changing expression of an identified protease by geneti(cid:173)
`cally modifying the host cell or organism to reduce the
`expression of the upregulated protease. In a further embodi(cid:173)
`ment, the process comprises changing the expression of at
`least a second identified protease in the modified cell to
`provide a double protease modified cell. In another embodi(cid:173)
`ment, the process further comprises: iv) expressing the
`recombinant protein or peptide, or a related protein or
`peptide, in a protease modified cell. In another embodiment,
`the process further comprises analyzing a second genetic
`profile of the protease modified cell to identify one or more
`additional gene products that are differentially expressed in
`the modified cell.
`
`[0034]
`In another embodiment, a process is provided for
`improving the expression of a recombinant protein or pep(cid:173)
`tide comprising: i) expressing the recombinant protein or
`peptide in a host cell; ii) analyzing a genetic profile of the
`cell and identifying at least one up-regulated folding modu(cid:173)
`lator (FM) that is up-regulated after overexpression of the
`recombinant protein or peptide; and iii) changing expression
`of at least one identified folding modulator by genetically
`modifying the cell to provide a FM modified cell. In a further
`embodiment, the process comprises changing the expression
`of at least a second identified folding modulator in the
`modified cell to provide a double FM modified cell. In
`another embodiment, the process further comprises: iv)
`expressing the recombinant protein or peptide, or a related
`protein or peptide, in a FM modified cell. In another embodi(cid:173)
`ment, the process further comprises analyzing a second
`genetic profile of the FM modified cell to identify one or
`more additional gene products
`that are differentially
`expressed in the modified cell.
`
`[0035] The term "genetic profile" as used herein is meant
`to include an analysis of genes in a genome, mRNA tran(cid:173)
`scribed from genes in the genome ( or the equivalent cDNA),
`transcription products that have been modified by a cell such
`as splice variants of genes in eukaryotic systems, or proteins
`or peptides translated from genes in a genome, including
`proteins that are modified by the cell or translated from
`splice variants of mRNA translated from the genome. A
`genetic profile is meant to include more than one gene or
`gene product, and typically includes a group of at least 5, 10,
`50, 100 or more genes or gene products that are analyzed.
`
`[0036]
`In one embodiment, the genetic profile analyzed
`can be a transcriptome profile, i.e. a profile of the transcrip-
`
`tion products of genes from the genome. The process can
`include analyzing the transcriptome profile using a microar(cid:173)
`ray or equivalent technology. In this embodiment, the
`microarray can include binding partners to at least a portion
`of the transcriptome of the host cell, and typically includes
`samples from binding partners to gene products of at least
`50% of the genome of the organism. More typically, the
`microarray includes samples from at least 80%, 90%, 95%,
`98%, 99% or 100% of the binding partners to gene products
`in the genome of the host cell.
`
`[0037]
`In a separate embodiment, the microarray can
`include a selected subset of binding partners to genes or gene
`products which represent classes of products that are
`affected by the recombinant protein expression. Nonlimiting
`examples include putative or known proteases,