1111111111111111 IIIIII IIIII 11111 1111111111 lllll 111111111111111 1111111111 1111111111 11111111
`US 20080269070Al
`
`c19) United States
`c12) Patent Application Publication
`Ramseier et al.
`
`c10) Pub. No.: US 2008/0269070 Al
`Oct. 30, 2008
`(43) Pub. Date:
`
`(54) METHOD FOR RAPIDLY SCREENING
`MICROBIAL HOSTS TO IDENTIFY CERTAIN
`STRAINS WITH IMPROVED YIELD AND/OR
`QUALITY IN THE EXPRESSION OF
`HETEROLOGOUS PROTEINS
`
`(75)
`
`Inventors:
`
`Thomas M. Ramseier, Newton,
`MA (US); Russell J. Coleman, San
`Diego, CA (US); Jane C.
`Schneider, San Diego, CA (US);
`Charles D. Hershberger, Poway,
`CA (US)
`
`Correspondence Address:
`Alston & Bird LLP
`Dow Global Technologies, Inc.
`Bank Of America Plaza, 101 South Tryon Street,
`Suite 4000
`Charlotte, NC 28280-4000 (US)
`
`(73) Assignee:
`
`Dow Global Technologies, Inc.,
`Midland, MI (US)
`
`(21) Appl. No.:
`
`12/109,554
`
`(22) Filed:
`
`Apr. 25, 2008
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/914,361, filed on Apr.
`27, 2007.
`Publication Classification
`
`(51)
`
`Int. Cl.
`C40B 30/00
`(2006.01)
`C40B 40102
`(2006.01)
`(52) U.S. Cl. ............................................... 506/14; 506/7
`ABSTRACT
`(57)
`
`The present invention provides an array for rapidly identify(cid:173)
`ing a host cell population capable of producing heterologous
`protein with improved yield and/or quality. The array com(cid:173)
`prises one or more host cell populations that have been geneti(cid:173)
`cally 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,
`or both. One or more of the strains in the array may express
`the heterologous protein of interest in a periplasm compart(cid:173)
`ment, or may secrete the heterologous protein extracellularly
`through an outer cell wall. The strain arrays are useful for
`screening for 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.
`
`Me I(Boos)
`
`RXF05464 pyrF
`
`Stem Loop
`
`srfl site
`
`lacPromottfr (-35 and -10)
`pUC ori edges uncertai
`
`Cm R
`
`pDOW1261-2
`84321:p
`
`tetR
`
`tetA
`
`Xiv If:ml)
`
`

`

`Patent Application Publication
`
`Oct. 30, 2008 Sheet 1 of 3
`
`US 2008/0269070 Al
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`
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`
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`
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`
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`
`75
`
`100
`150
`250
`
`

`

`US 2008/0269070 Al
`
`Oct. 30, 2008
`
`1
`
`METHOD FOR RAPIDLY SCREENING
`MICROBIAL HOSTS TO IDENTIFY CERTAIN
`STRAINS WITH IMPROVED YIELD AND/OR
`QUALITY IN THE EXPRESSION OF
`HETEROLOGOUS PROTEINS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims the benefit of U.S. Provi(cid:173)
`sionalApplicationNo. 60/914,361, filedApr. 27, 2007, which
`is hereby incorporated in its entirety by reference herein.
`
`REFERENCE TO A SEQUENCE LISTING
`SUBMITTED AS A TEXT FILE VIA EFS-WEB
`
`[0002] The official copy of the sequence listing is submitted
`concurrently with the specification as a text file via EFS-Web,
`in compliance with the American Standard Code for Infor(cid:173)
`mation Interchange (ASCII), with a file name of 342528_
`SequenceListing.txt, a creation date of Apr. 21, 2008 and a
`size of216 KB. The sequence listing filed via EFS-Web is part
`of the specification and is herein incorporated by reference in
`its entirety.
`
`FIELD OF THE INVENTION
`
`[0003] 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
`
`[0004] 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 pro(cid:173)
`duced 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 inefficiently
`achieved using known methods.
`[0005] Numerous attempts have been developed to increase
`production of properly folded proteins in recombinant sys(cid:173)
`tems. For example, investigators have changed fermentation
`conditions (Schein (1989) Bio/Technology, 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.
`[0006] 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).
`However, the majority of research has focused on the secre(cid:173)
`tion of exogenous proteins with a Sec-type secretion system.
`[0007] 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. 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 O 121 352.
`[0008] 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 pro(cid:173)
`tein of interest and thus reduce the final yield. There is no
`single factor that will improve the production of all heterolo(cid:173)
`gous proteins. As a result, there is a need in the art for iden(cid:173)
`tifying improved large-scale expression systems capable of
`secreting and properly processing recombinant polypeptides
`to produce transgenic proteins in properly processed form.
`
`SUMMARY OF THE INVENTION
`
`[0009] 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. 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 inter(cid:173)
`est, but includes yield, quality, activity, and the like.
`[001 OJ
`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
`expression of the polypeptide ofinterest. 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 fold(cid:173)
`ing modulator is selected from the list in Table 1.
`[0011]
`In another embodiment, one or more of the host cell
`populations has been genetically modified to decrease 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.
`[0012]
`In one embodiment, nucleotide sequences encoding
`the proteins of interest are operably linked to a P. jluorescens
`Sec system secretion signal as described herein. One or more
`of the strains in the array may express the heterologous pro(cid:173)
`tein of interest in a periplasm compartment. In certain
`embodiments, one or more strains may also secrete the het(cid:173)
`erologous protein extracellularly through an outer cell wall.
`[0013] Host cells include eukaryotic cells, including yeast
`cells, insect cells, mammalian cells, plant cells, etc., and
`prokaryotic cells, including bacterial cells such as P. jluore(cid:173)
`scens, E. coli, and the like.
`
`

`

`US 2008/0269070 Al
`
`Oct. 30, 2008
`
`2
`
`[0014] As indicated, the library ofhost cell populations can
`be rapidly screened to identify certain strain( s) with improved
`yield and/or quality ofheterologously expressed protein. The
`strain arrays are useful for screening for improved expression
`of any protein of interest, including therapeutic proteins, hor(cid:173)
`mones, a growth factors, extracellular receptors or ligands,
`proteases, kinases, blood proteins, chemokines, cytokines,
`antibodies and the like.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0015] FIG. lA depicts plasmid pDOW1261-2 used for
`engineering genomic deletion in P. jluorescens. FIG. lB is a
`schematic drawing of the constructions of a gene X deletion.
`[0016] FIG. 2 is a Western blot analysis of soluble cells
`fractions prepared at O and 24 hours post-induction (IO and
`124, respectively) inllprcl, lldegP2, llLa2 and the grpEdnaKJ
`co-expression strains (Example 6). The top arrows point to
`the fully assembled monoclonal antibody in the co-expressed
`strains but not in the control (DC440). r=recombinant;
`n-r=nonrecombinant.
`
`DETAILED DESCRIPTION
`
`[0017] The present inventions now will be described more
`fully hereinafter with reference to the accompanying draw(cid:173)
`ings, in which some, but not all embodiments of the invention
`are shown. Indeed, these inventions may be embodied in
`many different forms and should not be construed as limited
`to the embodiments set forth herein; rather, these embodi(cid:173)
`ments are provided so that this disclosure will satisfy appli(cid:173)
`cable legal requirements.
`[0018] 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.
`[0019] 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. Although spe(cid:173)
`cific terms are employed herein, they are used in a generic and
`descriptive sense only and not for purposes of limitation.
`
`Overview
`
`[0020] Compositions and methods for identifying an opti(cid:173)
`mal host strain, e.g, a Pseudomonas jluorescens host strain,
`for producing high levels of properly processed heterologous
`polypeptides in a host cell are provided. In particular, a library
`( or "array") ofhost strains is provided, wherein each strain ( or
`"population of host cells") in the library has been genetically
`modified to modulate the expression of one or more target
`genes in the host cell. An "optimal host strain" can be iden(cid:173)
`tified or selected based on the quantity, quality, and/or loca(cid:173)
`tion of the expressed protein of interest compared to other
`populations of phenotypically distinct host cells in the array.
`Thus, an optimal host strain is the strain that produces the
`polypeptide of interest according to a desired specification.
`While the desired specification will vary depending on the
`polypeptide being produced, the specification includes the
`quality and/or quantity of protein, whether the protein is
`sequestered or secreted, protein folding, and the like.
`[0021]
`"Heterologous," "heterologously expressed," or
`"recombinant" generally refers to a gene or protein that is not
`endogenous to the host cell or is not endogenous to the loca-
`
`tion in the native genome in which it is present, and has been
`added to the cell by infection, transfection, microinjection,
`electroporation, microprojection, or the like.
`[0022] One or more of the host cell populations in the array
`is modified to modulate the expression of one or more target
`genes in the host cell. By "target gene" is intended a gene that
`affects heterologous protein production in a host cell. Target
`genes that affect heterologous protein production include
`genes encoding proteins that modulate expression, activity,
`solubility,
`translocation, proteolytic degradation and/or
`cleavage of the heterologous protein. For example, a target
`gene may encode at least one of a host cell protease, a protein
`folding modulator, a transcription factor, a translation factor,
`a secretion modulator, or any other protein involved in the
`proper transcription, translation, processing, and/or translo(cid:173)
`cation of a heterologous protein of interest. A "target protein"
`refers to the protein or polypeptide resulting from expression
`of the target gene. Expression and/or activity of a target gene
`or genes is increased or decreased, depending on the function
`of the target gene or protein. For example, expression of one
`or more host cell proteases may be decreased, whereas
`expression of one or more protein folding modulators may be
`increased.
`[0023] The arrays described herein are useful for rapidly
`identifying an optimal host cell for production of a heterolo(cid:173)
`gous protein or peptide of interest. Heterologous protein pro(cid:173)
`duction often leads to the formation of insoluble or improp(cid:173)
`erly folded proteins, which are difficult to recover and may be
`inactive. Furthermore, the presence of specific host cell pro(cid:173)
`teases may degrade the protein of interest and thus reduce the
`final yield. There is no single host cell population that will
`optimally produce all polypeptides or proteins of interest.
`Thus, using the compositions and methods of the invention,
`an optimal host cell can be rapidly and efficiently identified
`from the library of modified cell populations. The optimal
`host strain can then be used to produce sufficient amounts of
`the protein of interest or for commercial production. Like(cid:173)
`wise, a host strain can be modified for expression of the
`protein of interest based on the optimal host strain.
`[0024]
`In one embodiment, the method includes obtaining
`an array comprising at least a first and a second population of
`P. jluorescens cells, wherein each population is selected from
`the group consisting of (i) a population of P. jluorescens cells
`that has been genetically modified to reduce the expression of
`at least target gene involved in protein degradation; (ii) a
`population of P. jluorescens cells that has been genetically
`modified to increase the expression of at least one target gene
`involved in protein production; and, (iii) a population of P.
`jluorescens cells that has been genetically modified to reduce
`the expression of at least one target gene involved in protein
`degradation and to increase the expression of at least target
`gene involved in protein production; introducing into at least
`one cell of each population an expression construct compris(cid:173)
`ing at least one gene encoding at least one heterologous
`protein of interest; maintaining said cells under conditions
`sufficient for the expression of said protein of interest in at
`least one population of cells; and selecting the optimal popu(cid:173)
`lation of cells in which the heterologous protein of interest is
`produced; wherein each population in the array is non-iden(cid:173)
`tical and wherein each population is physically separate one
`from another; wherein the heterologous protein of interest
`exhibits one or more ofimproved expression, improved activ(cid:173)
`ity, improved solubility, improved translocation, or reduced
`
`

`

`US 2008/0269070 Al
`
`Oct. 30, 2008
`
`3
`
`proteolytic degradation or cleavage in the optimal population
`of cells compared to other populations in the array.
`[0025] The array may further comprise a population of host
`cells ( e.g., P. jluorescens host cells) that has not been geneti(cid:173)
`cally modified to alter the expression of a host cell protease or
`a protein folding modulator. This population may be a wild(cid:173)
`type strain, or may be a strain that has been genetically modi(cid:173)
`fied to alter the expression of or more genes not involved in
`protein production, processing, or translocation ( e.g., may be
`genetically modified to express, for example, a selectable
`marker gene).
`In one embodiment, each population of P. jluore(cid:173)
`[0026]
`scens host cells is phenotypically distinct (i.e., "non-identi(cid:173)
`cal") one from another. By "phenotypically distinct" is
`intended that each population produces a measurably differ(cid:173)
`ent amount of one or more target proteins. In this embodi(cid:173)
`ment, each strain has been genetically modified to alter the
`expression of one or more different target genes. Where the
`expression of more than one target gene is modulated in a
`population of host cells, then the combination of target genes
`is phenotypically distinct from other populations in the
`library. An array comprising a plurality of phenotypically
`distinct populations of host cells according to the present
`invention is one that provides a diverse population from
`which to select one or more strains useful for producing a
`heterologous protein or peptide of interest. It will be under(cid:173)
`stood by one of skill in the art that such an array may also
`comprise replicates ( e.g., duplicates, triplicates, etc.) of any
`one or more populations of host cells.
`
`Arrays
`
`[0027] Provided herein is an array of host cell populations
`(i.e. "strain array") which can be rapidly screened to identify
`certain strain(s) with improved yield and/or quality ofheter(cid:173)
`ologous protein. As used herein, the term "strain array" refers
`to a plurality of addressed or addressable locations ( e.g.,
`wells, such as deep well or microwells). The location of each
`of the microwells or groups of
`microwells in the array is typically known, so as to allow for
`identification of the optimal host cell for expression of the
`heterologous protein of interest.
`[0028] The strain array comprises a plurality of phenotypi(cid:173)
`cally distinct host strains. The arrays may be low-density
`arrays or high-density arrays and may contain about 2 or
`more, about 4 or more, about 8 or more, about 12 or more,
`about 16 or more, about 20 or more, about 24 or more, about
`32 or more, about 40 or more, about 48 or more, about 64 or
`more, about 72 or more, about 80 or more, about 96 or more,
`about 192 or more, about 384 or more host cell populations.
`[0029] The host cell populations of the invention can be
`maintained and/or screened in a multi-well or deep well ves(cid:173)
`sel. The vessel may contain any desired number of wells,
`however, a miniaturized cell culture microarray platform is
`useful for screening each population ofhost cells individually
`and simultaneously using minimal reagents and a relatively
`small number of cells. A typical multi-well, microtiter vessel
`useful in this assay is a multi-well plate including, without
`limitation, 10-well plates, 28-well plates, 96-well plates, 384-
`well plates, and plates having greater than 384 wells. Alter(cid:173)
`natively, an array of tubes, holders, cartridges, minitubes,
`microfuge tubes, cryovials, square well plates tubes, plates,
`slants, or culture flasks may also be used, depending on the
`volume desired.
`
`[0030] The vessel may be made of any material suitable for
`culturing and/or screening a host cell of interest, e.g.,
`Pseudomonas. For example, the vessel can be a material that
`can be easily sterilized such as plastic or other artificial poly(cid:173)
`mer material, so long as the material is biocompatible. Any
`number of materials can be used, including, but not limited to,
`polystyrene; polypropylene; polyvinyl compounds (e.g.
`polyvinylchloride ); polycarbonate (PVC); polytetrafluoroet(cid:173)
`hylene (PTFE); polyglycolic acid (PGA); cellulose; glass,
`fluoropolymers, fluorinated ethylene propylene, polyvi(cid:173)
`nylidene, polydimethylsiloxane, silicon, and the like.
`[0031] Automated transformation of cells and automated
`colony pickers will facilitate rapid screening of desired cells.
`The arrays may be created and/or screened using a spotter
`device (e.g., automated robotic devices) as known in the art.
`
`Target Genes
`
`[0032] The strain array of the present invention comprises a
`plurality of phenotypically and genotypically distinct host
`cell populations, wherein each population in the array has
`been genetically modified to modulate the expression of one
`or more target genes in the host cell. By "target gene" is
`intended a gene that affects heterologous protein production
`in a host cell. A target gene may encode a host cell protease or
`an endogenous or exogenous protein folding modulator, tran(cid:173)
`scription factor, translation factor, secretion modulator, or
`any other gene involved in the proper expression, processing,
`and/or translocation of a heterologous protein of interest. A
`"target protein" refers to the protein or polypeptide resulting
`from expression of the target gene. Expression and/or activity
`of a target gene or genes is increased or decreased, depending
`on the function of the target gene or protein. A target gene can
`be endogenous to the host cell, or can be a gene that is
`heterologously expressed in each of the host cell populations
`in the array.
`In one embodiment, the target gene or genes is at
`[0033]
`least one protein folding modulator, putative protein folding
`modulator, or a cofactor or subunit of a folding modulator. In
`some embodiments, the target gene or genes can be selected
`from a chaperone protein, a foldase, a peptidyl prolyl
`isomerase and a disulfide bond isomerase. In some embodi(cid:173)
`ments, the target gene or genes can be selected from htpG,
`cbpA, dnaJ, dnaK and fkbP. Exemplary protein folding
`modulators from P. jluorescens are listed in Table 1.
`In other embodiments, the target gene comprises at
`[0034]
`least one putative protease, a protease-like protein, or a cofac(cid:173)
`tor or subunit of a protease. For example, the target gene or
`genes can be a serine, threonine, cysteine, aspartic or metal(cid:173)
`lopeptidase. In one embodiment, the target gene or genes can
`be selected from hslV, hs!U, clpA, clpB and clpX. The target
`gene can also be a cofactor of a protease. Exemplary proteases
`from P. jluorescens are listed in Table 2. Proteases from a
`variety of organisms can be found in the MERO PS Peptidase
`Database maintained by the Welcome Trust Sanger Institute,
`Cambridge, UK (see the website address merops.sanger.ac.
`uk/).
`[0035] Protein Folding Modulators
`[0036] Another major obstacle in the production ofheter(cid:173)
`ologous proteins in host cells is that the cell often is not
`adequately equipped to produce either soluble or active pro(cid:173)
`tein. While the primary structure of a protein is defined by its
`amino acid sequence, the secondary structure is defined by
`the presence of alpha helices or beta sheets, and the ternary
`structure by covalent bonds between adjacent protein
`
`

`

`US 2008/0269070 Al
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`Oct. 30, 2008
`
`4
`
`stretches, such as disulfide bonds. When expressing heterolo(cid:173)
`gous proteins, particularly in large-scale production, the sec(cid:173)
`ondary and tertiary structure of the protein itself is of critical
`importance. Any significant change in protein structure can
`yield a functionally inactive molecule, or a protein with sig(cid:173)
`nificantly reduced biological activity. In many cases, a host
`cell expresses protein folding modulators (PFMs) that are
`necessary for proper production of active heterologous 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 cannot produce enough
`native protein folding modulator or modulators to process the
`heterologously-expressed protein.
`[0037]
`In certain expression systems, overproduction of
`heterologous proteins can be accompanied by their misfold(cid:173)
`ing 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 disaggregation.
`However, proteins processed to inclusion bodies can, in cer(cid:173)
`tain cases, be recovered through additional processing of the
`insoluble fraction. Proteins found in inclusion bodies typi(cid:173)
`cally have to be purified through multiple steps, including
`denaturation and renaturation. Typical renaturation processes
`for inclusion body targeted proteins involve attempts to dis(cid:173)
`solve the aggregate in concentrated denaturant and subse(cid:173)
`quent removal of the denaturant by dilution. Aggregates are
`frequently formed again in this stage. The additional process(cid:173)
`ing adds cost, there is no guarantee that the in vitro refolding
`will yield biologically active product, and the recovered pro(cid:173)
`teins can include large amounts of fragment impurities.
`[0038] The 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 fol(cid:173)
`dases, which accelerate rate-limiting steps along the folding
`pathway, has provided additional approaches to combat the
`problem ofinclusion body formation (see for e.g. Thomas JG
`et al. (1997) Appl Biochem Biotechnol 66: 197-238).
`[0039]
`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) Curr. Opin. Biotech.
`10:411-421 and references therein). The beneficial effect
`associated with an increase in the intracellular concentration
`of these chaperones appears highly dependent on the nature
`of the overproduced protein, and may not require overexpres(cid:173)
`sion of the same protein folding modulator(s) for all heter(cid:173)
`ologous proteins.
`[0040] Protein folding modulators, including chaperones,
`disulfide bond isomerases, and peptidyl-prolyl cis-trans
`isomerases (PPiases) are a class of proteins present in all cells
`which aid in the folding, unfolding and degradation of
`nascent polypeptides.
`[0041] Chaperones act by binding to nascent polypeptides,
`stabilizing them and allowing them to fold properly. Proteins
`possess both hydrophobic and hydrophilic residues, the
`former are usually exposed on the surface while the latter are
`buried within the structure where they interact with other
`hydrophilic residues rather than the water which surrounds
`the molecule. However in folding polypeptide chains, the
`hydrophilic residues are often exposed for some period of
`
`time as the protein exists in a partially folded or misfolded
`state. It is during this time when the forming polypeptides can
`become permanently misfolded or interact with other mis(cid:173)
`folded proteins and form large aggregates or inclusion bodies
`within the cell. Chaperones generally act by binding to the
`hydrophobic regions of the partially folded chains and pre(cid:173)
`venting them from misfolding completely or aggregating
`with other proteins. Chaperones can even bind to proteins in
`inclusion bodies and allow them to disaggregate (Ranson et.
`al. 1998). The GroES/EL, DnaKJ, Clp, Hsp90 and SecB
`families of folding modulators are all examples of proteins
`with chaperone like activity.
`[0042] Another important type of folding modulator is the
`disulfide bond isomerases. These proteins catalyze a very
`specific set of reactions to help folding polypeptides form the
`proper intra-protein disulfide bonds. Any protein that has
`more than two cysteines is at risk of forming disulfide bonds
`between the wrong residues. The disulfide bond formation
`family consists of the Dsb proteins which catalyze the forma(cid:173)
`tion of disulfide bonds in the non-reducing environment of the
`periplasm. When a periplasmic polypeptide misfolds disul(cid:173)
`fide bond isomerase, DsbC is capable of rearranging the
`disulfide bonds and allowing the protein to reform with the
`correct linkages.
`[0043] The pro line residue is unique among amino acids in
`that the peptidyl bond immediately preceding it can adopt
`either a cis or trans conformation. For all other amino acids
`this is not favored due to steric hindrance. Peptidyl-prolyl
`cis-trans isomerases (PPiases) catalyze the conversion of this
`bond from one form to the other. This isomerization may aid
`in protein folding, refolding, assembly of subunits and traf(cid:173)
`ficking in the cell (Dolinski, et. al. 1997).
`[0044]
`In addition to the general chaperones which seem to
`interact with proteins in a non-specific manner, there are also
`chaperones which aid in the folding of specific targets. These
`protein-specific chaperones form complexes with their tar(cid:173)
`gets, preventing aggregation and degradation and allowing
`time for them to assemble into multi-subunit structures. The
`PapD chaperone is one well known example of this type
`(Lombardo et. al. 1997).
`[0045] Folding modulators also include, for example,
`HSP70 proteins, HSPl 10/SSE proteins, HSP40 (DNAJ-re(cid:173)
`lated) proteins, GRPE-like proteins, HSP90 proteins, CPN60
`and CPNl 0 proteins, Cytosolic chaperoning, HS Pl 00 pro(cid:173)
`teins, Small HSPs, Calnexin and calreticulin, PDI and thiore(cid:173)
`doxin-related proteins, Peptidyl-prolyl isomerases, Cyclo(cid:173)
`philin PPiases, FK-506 binding proteins, Parvulin PPiases,
`Individual chaperoning, Protein specific chaperones, or
`intramolecular chaperones. Folding modulators are generally
`described in "Guidebook to Molecular Chaperones