Choice of Cellular Protein Expression System

David Gray1, Shyam Subramanian2

1 Chiron Corporation, Emeryville, California, 2 Merck Research Laboratories, West Point, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.16
DOI:  10.1002/0471140864.ps0516s20
Online Posting Date:  May, 2001
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Abstract

Recombinant protein expression has become a standard laboratory tool, and a wide variety of systems and techniques are now in use. Because there are so many systems to choose from, the investigator has to be careful to use the combination that will give the best results for the protein being studied. This overview unit discusses expression and production choices, including post‐translational modifications (e.g., glycosylation, acylation, sulfation, and removal of N‐terminal methionine), in vivo and in vitro folding, and influence of downstream elements on expression.

     
 
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Table of Contents

  • Background
  • Expression Systems
  • Post‐Translational Modifications and Expression System Choice
  • Production of Soluble Proteins by Host Cells
  • Protein‐Based Features
  • Special Considerations
  • Conclusions
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

Literature Cited
   Abeijon, C., Mandon, E.C., and Hirschberg, C.B. 1997. Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus. Trends Biochem. Sci. 22:203‐207.
   Ailor, E. and Betenbaugh, M.J. 1999. Modifying secretion and posttranslational processing in insect cells. Insect cell secretion and posttranslational modifications. Curr. Opin. Biotechnol. 10:142‐145.
   Allen, S. and Bulleid, N.J. 1997. Calnexin and calreticulin bind to enzymically active tissue‐type plasminogen activator during biosynthesis and are not required for folding to the native conformation. Biochem. J. 328:113‐119.
   Allen, S., Naim, H.Y., and Bulleid, N.J. 1995. Intracellular folding of tissue‐type plasminogen activator. Effects of disulfide bond formation on N‐linked glycosylation and secretion. J. Biol. Chem. 270:4797‐4804.
   Altmann, F., Schwihla, H., Staudacher, E., Glossl, J., and Marz, L. 1995. Insect cells contain an unusual, membrane‐bound β‐N‐acetylglucosaminidase probably involved in the processing of protein N‐glycans. J. Biol. Chem. 270:17344‐17349.
   Aruffo, A. 1998. Transient expression of proteins using COS cells. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.13.1‐16.13.7. John Wiley & Sons, New York.
   Babbitt, P.C., West, B.L., Beuchter, D.D., Kuntz, I.D., and Kenyon, G.L. 1990. Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme. Bio/Technology 8:945‐949.
   Ballou, C.E. 1990. Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 185:440‐469.
   Ballou, L., Cohen, R.E., and Ballou, C.E. 1980. Saccharomyces cerevisiae mutants that make mannoproteins with a truncated carbohydrate outer chain. J. Biol. Chem. 255:5986‐5991.
   Barr, P.J. 1992. Expression of foreign genes in yeast. In Transgenesis: Applications of Gene Transfer (J.A.H. Murray, ed.) pp. 55‐79. John Wiley & Sons, New York.
   Barr, P.J., Gibson, H.L., Enea, V., Arnot, D.E., Hollingdale, M.R., and Nussenzweig, V. 1987a. Expression in yeast of a Plasmodium vivax antigen of potential use in a human malaria vaccine. J. Exp. Med. 165:1160‐1171.
   Barr, P.J., Gibson, H.L., Lee‐Ng, C.T., Sabin, E.A., Power, M.D., Brake, A.J., and Shuster, J.R. 1987b. Heterologous gene expression in the yeast Saccharomyces cerevisiae. In Industrial Yeast Genetics, Vol. 5 (M. Korhola and H. Nevalainen, eds.) pp. 139‐148. Foundation for Biotechnical and Industrial Fermentation Research, Helsinki.
   Barr, P.J., Cousens, L.S., Lee‐Ng, C.T., Medina‐Selby, A., Masiarz, F.R., Hallewell, R.A., Chamberlain, S., Bradley, J., Steimer, K.S., Esch, F., and Baird, A. 1988. Expression and processing of biologically active fibroblast growth factors in yeast Saccharomyces cerevisiae. J. Biol. Chem. 263:16471‐16478.
   Barr, P.J., Sabin, E.A., Ng, C.T.L., Lansberg, K.E., Steimer, K.S., Bathurst, I.C., and Schuster, J.R. 1991. Ubiquitin fusion approach to heterologous gene expression in yeast: High‐level production of amino‐terminal authentic proteins. In Expression Systems and Processes for rDNA Products (R.T. Hatch, C. Goochee, A. Moreiro, and A. Yoir, eds.) pp. 51‐64. American Chemical Society, Washington, D.C.
   Bathurst, I.C., Chester, N., Gibson, H.L., Dennis, A.F., Steimer, K.S., and Barr, P.J. 1989. N‐myristylation of the human immunodeficiency virus type 1 gag polyprotein precursor in Saccharomyces cerevisiae. J. Virol. 63:3176‐3179.
   Beckmann, R.P., Mizzen, L.E., and Welch, W.J. 1990. Interaction of Hsp70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248:850‐854.
   Bhatia, P.K. and Mukhopadhyay, A. 1998. Protein glycosylation: Implications for in vivo functions and therapeutic applications. Adv. Biochem. Eng. Biotechnol. 64:155‐201.
   Bogosian, G., Violand, B.N., Dorward‐King, E.J., Workman, W.E., Jung, P.E., and Kane, J.F. 1989. Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J. Biol. Chem. 264:531‐539.
   Boulain, J.C., Charbit, A., and Hofnung, M. 1986. Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12. Mol. Gen. Genet. 205:339‐348.
   Bowden, G.A., Paredes, A.M., and Georgiou, G. 1991. Structure and morphology of protein inclusion bodies in Escherichia coli. Bio/Technology 9:725‐729.
   Brady, R.O., Murray, G.J., and Barton, N.W. 1994. Modifying exogenous glucocerebrosidase for effective replacement therapy in Gaucher disease. J. Inherited Metab. Dis. 17:510‐519.
   Burgess, A.W., Begley, C.G., Johnson, G.R., Lopez, A.F., Williamson, D.J., Mermod, J.J., Simpson, R.J., Schmitz, A., and DeLamarter, J.F. 1987. Purification and properties of bacterially synthesized human granulocyte‐macrophage colony stimulating factor. Blood 69:43‐51.
   Canaud, B., Polito‐Bouloux, C., Garred, L.J., Rivory, J.P., Donnadieu, P., Taib, J., Florence, P., and Mion, C. 1990. Recombinant human erythropoietin: 18 months' experience in hemodialysis patients. Am. J. Kidney Dis. 15:169‐175.
   Chapot, M.P., Eshdat, Y., Marullo, S., Guilett, J.G., Charbit, A., Strosberg, A.D., Delavier‐Klutchko, C. 1990. Localization and characterization of three different beta‐adrenergic receptors expressed in Escherichia coli. Eur. J. Biochem. 187:137‐144.
   Clarke, S. 1992. Protein isoprenylation and methylation at carboxyterminal cysteine residues. Annu. Rev. Biochem. 61:355‐386.
   Davidson, D.J. and Castellino, F.J. 1991a. Asparagine‐linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine‐289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB‐SF‐21AE) cells. Biochemistry 30:6165‐6174.
   Davidson, D.J. and Castellino, F.J. 1991b. Structures of the asparagine‐289‐linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD‐MBO503). Biochemistry 30:6689‐6696.
   Davidson, H.W., Rhodes, C.J., and Hutton, J.C. 1988. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site specific endopeptidases. Nature 333:93‐96.
   Davidson, D.J., Fraser, M.J., and Castellino, F.J. 1990. Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells. Biochemistry 29:5584‐5590.
   Davie, J.R. 1997. Nuclear matrix, dynamic histone acetylation and transcriptionally active chromatin. Mol. Biol. Rep. 24:197‐207.
   Dean, N. 1999. Asparagine‐linked glycosylation in the yeast Golgi. Biochim. Biophys. Acta 1426:309‐322.
   Delorme, E., Lorenzini, T., Giffin, J., Martin, F., Jacobsen, F., Boone, T., and Elliot, S. 1992. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31:9871‐9876.
   Donaldson, M., Wood, H.A., Kulakosky, P.C., and Shuler, M.L. 1999. Use of mannosamine for inducing the addition of outer arm N‐acetylglucosamine onto N‐linked oligosaccharides of recombinant proteins in insect cells. Biotechnol. Prog. 15:168‐173.
   Dorner, A.J. and Kaufman, R.J. 1994. The levels of endoplasmic reticulum proteins and ATP affect folding and secretion of selective proteins. Biologicals 22:103‐112.
   Duronio, R.J., Jackson‐Machelski, E., Heuckeroth, R.O., Olins, P.O., Devine, C.S., Yonemoto, W., Slice, L.W., Taylor, S.S., and Gordon, J.I. 1990. Protein N‐myristoylation in Escherichia coli: Reconstitution of a eukaryotic protein modification in bacteria. Proc. Natl. Acad. Sci. U.S.A. 87:1506‐1510.
   Ellis, R.J. and Hartl, F.U. 1996. Protein folding in the cell: Competing models of chaperonin function. FASEB J. 10:20‐26.
   Ewalt, K.L., Hendrick, J.P., Houry, W.A., and Hartl, F.U. 1997. In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell 90:491‐500.
   Faber, K.N., Harder, W., Geert, A.B., and Veenhuis, M. 1995. Review: Methylotrophic yeasts as factories for the production of foreign proteins. Yeast 11:1331‐1344.
   Fallaux, F.J., Bout, A., van der Velde, I., van den Wollenberg, D.J., Hehir, K.M., Keegan, J., Auger, C., Cramer, S.J., van Ormondt, H., van der Eb, A.J., Valerio, D., and Hoeben, R.C. 1998. New helper cells and matched early region 1‐deleted adenovirus vectors prevent generation of replication‐competent adenoviruses. Hum. Gene Ther. 9:1909‐1917.
   Fann, C.H., Guarna, M.M., Kilburn, D.G., and Piret, J.M. 1999. Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells. Biotechnol. Bioeng. 63:464‐472.
   Farrell, E.R. and Keshishian, H. 1999. Laser ablation of persistent twist cells in Drosophila: Muscle precursor fate is not segmentally restricted. Development 126:273‐280.
   Fleer, R., Chen, X.J., Amellal, N., Yeh, P., Fournier, A., Guinet, F., Gault, N., Faucher, D., Folliard, F., Fukuhara, H., et al. 1991. High‐level secretion of correctly processed recombinant human interleukin‐1β in Kluyveromyces lactis. Gene 107:285‐295.
   Foster, D.C., Rudinski, M.S., Schach, B.G., Berkner, K.L., Kumar, A.A., Hagen, F.S., Sprecher, C.A., Insley, M.Y., and Davie, E.W. 1987. Propeptide of human protein C is necessary for γ‐carboxylation. Biochemistry 26:7003‐7011.
   Frydman, J., Nemessgern, E., Ohtsuka, K., and Hartl, F.U. 1994. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370:111‐117.
   Fukuda, M.N., Sasaki, H., Lopez, L., and Fukuda, M. 1989. Survival of recombinant erythropoietin in the circulation: The role of carbohydrates. Blood 73:84‐89.
   Fukushige, S. and Sauer, B. 1992. Genomic targeting with a positive‐selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 89:7905‐7909.
   Galili, U. 1993. Evolution and pathophysiology of the human natural anti‐α‐galactosyl IgG (anti‐Gal) antibody. Springer Semin. Immunopathol. 15:155‐171.
   Garel, J.‐R. 1992. Folding of large proteins: Multidomain and multisubunit proteins. In Protein Folding (T.E. Creighton, ed.) pp. 405‐454. W.H. Freeman, New York.
   Gellissen, G. 1994. Heterologous gene expression in C1 compound‐utilizing yeasts. Bioprocess Technol. 19:787‐796.
   Gellissen, G., Weydemann, U., Strasser, A.W., Piontek, M., Janowicz, Z.A., and Hollenberg, C.P. 1992. Progress in developing methylotrophic yeasts as expression systems. Trends Biotechnol. 10:413‐417.
   Gemmill, T.R. and Trimble, R.B. 1999. Overview of N‐ and O‐linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426:227‐237.
   George‐Nascimento, C., Gyenes, A., Halloran, M., Merryweather, J., Valenzuela, P., Steimer, K.S., Masiarz, F.R., and Randolph, A. 1988. Characterization of recombinant human epidermal growth factor produced in yeast. Biochemistry 27:797‐802.
   Goeddel, D.V. 1990. Systems for heterologous gene expression. Methods Enzymol. 185:3‐7.
   Goochee, C.F. and Monica, T. 1990. Environmental effects on protein glycosylation. Bio/Technology 8:421‐427.
   Goochee, C.F., Gramer, M.J., Andersen, D.C., Bahr, J.B., and Rasmussen, J.R. 1991. The oligosaccharides of glycoproteins: Factors affecting their synthesis and their influence on glycoprotein properties. In Frontiers in Bioprocessing II (S.K. Sidkar, M. Bier, and P. Todd, eds.) pp. 199‐240. American Chemical Society, Washington, D.C.
   Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G., and Conradt, H.S. 1995. Construction of stable BHK‐21 cells coexpressing human secretory glycoproteins and human Gal(β1‐4)GlcNAc‐R α2,6‐sialyltransferase. α2,6‐linked NeuAc is preferentially attached to the Gal(β1‐4)GlcNAc(β1‐2)Man(α1‐3)‐branch of diantennary oligosaccharides from secreted recombinant β‐trace protein. Eur. J. Biochem. 232:718‐725.
   Gramer, M.J. and Goochee, C.F. 1993. Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant. Biotechnol. Prog. 9:366‐373.
   Gramer, M.J. and Goochee, C.F. 1994. Glycosidase activities of the 293 and NS0 cell lines, and of an antibody‐producing hybridoma cell line. Biotechnol. Bioeng. 43:423‐428.
   Gramer, M.J., Goochee, C.F., Chock, V.Y., Brousseau, D.T., and Sliwkowski, M.B. 1995. Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Bio/Technology 13:692‐698.
   Grammatikos, S.I., Valley, U., Nimtz, M., Conradt, H.S., and Wagner, R. 1998. Intracellular UDP‐N‐acetylhexosamine pool affects N‐glycan complexity: A mechanism of ammonium action on protein glycosylation. Biotechnol. Prog. 14:410‐419.
   Gu, X. and Wang, D.I.C. 1998. Improvement of interferon‐γ sialylation in Chinese hamster ovary cell culture by feeding of N‐acetylmannosamine. Biotechnol. Bioeng. 58:642‐648.
   Hallewell, R.A., Mills, R., Tekamp‐Olson, P., Blacher, R., Rosenberg, S., Otting, F., Masiarz, F.R., and Scandella, C.J. 1987. Amino terminal acetylaton of authentic human Cu, Zn superoxide dismutase produced in yeast. Bio/Technology 5:363‐366.
   Hampsey, D.M., Das, G., and Sherman, F. 1986. Amino acid replacements in yeast iso‐1‐cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c. J. Biol. Chem. 261:3259‐3271.
   Harmsen, M.M., Bruyne, M.I., Raue, H.A., and Maat, J. 1996. Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast. Appl. Microbiol. Biotechnol. 46:365‐370.
   Hart, R.A., Lester, P.M., Reifsnyder, D.H., Ogez, J.R., and Builder, S.E. 1994. Large scale, in situ isolation of periplasmic IGF‐I from E. coli. Bio/Technology 12:1113‐1117.
   Hartl, F.U. 1996. Molecular chaperones in cellular protein folding. Nature 381:571‐580.
   Helenius, A., Trombetta, E.S., Hebert, D.N., and Simons, J.F. 1997. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7:193‐200.
   Hendrick, J.P., Langer, T., Davis, T.A., Hartl, F.U., and Weidmann, M. 1993. Control of folding and membrane translocation by binding of the chaperone DNAJ to nascent polypeptides. Proc. Natl. Acad. Sci. U.S.A. 90:10216‐10220.
   Herscovics, A. and Orlean, P. 1993. Glycoprotein biosynthesis in yeast. FASEB J. 7:540‐550.
   Higuchi, M., Oh‐eda, M., Kuboniwa, H., Tomonoh, K., Shimonaka, Y., and Ochi, N. 1992. Role of sugar chains in the expression of the biological activity of human erythropoietin. J. Biol. Chem. 267:7703‐7709.
   Hodgson, J. 1993. Expression systems: A user's guide. Bio/Technology 11:887‐888, 890, 893.
   Hollister, J.R., Shaper, J.H., and Jarvis, D.L. 1998. Stable expression of mammalian β 1,4‐galactosyltransferase extends the N‐glycosylation pathway in insect cells. Glycobiology 8:473‐480.
   Hooker, A.D., Green, N.H., Baines, A.J., Bull, A.T., Jenkins, N., Strange, P.G., and James, D.C. 1999. Constraints on the transport and glycosylation of recombinant IFN‐γ in Chinese hamster ovary cells and insect cells. Biotechnol. Bioeng. 63:559‐572.
   Hotchkiss, A., Refino, C.J., Leonard, C.K., O'Connor, J.V., Crowley, C., McCabe, J., Tate, K., Nakamura, G., Powers, D., Levinson, A., et al. 1988. The influence of carbohydrate structure on the clearance of recombinant tissue‐type plasminogen activator. Thromb. Haemost. 60:255‐261.
   Hounsell, E.F., Davies, M.J., and Renouf, D.V. 1996. O‐linked protein glycosylation structure and function. Glycoconjugate J. 13:19‐26.
   Howard, S.C., Wittwer, A.J., and Welply, J.K. 1991. Oligosaccharides at each glycosylation site make structure‐dependent contributions to biological properties of human tissue plasminogen activator. Glycobiology 1:411‐418.
   Hsu, T.A., Takahashi, N., Tsukamoto, Y., Kato, K., Shimada, I., Masuda, K., Whiteley, E.M., Fan, J.Q., Lee, Y.C., and Betenbaugh, M.J. 1997. Differential N‐glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J. Biol. Chem. 272:9062‐9070.
   Hwang, C., Lodish, H.F., and Sinskey, A.J. 1995. Measurement of glutathione redox state in cytosol and secretory pathway of cultured cells. Methods Enzymol. 251:212‐221.
   Imai, N., Higuchi, M., Kawamura, A., Tomonoh, K., Oh‐eda, M., Fujiwara, M., Shimonaka, Y., and Ochi, N. 1990. Physicochemical and biological characterization of asialoerythropoietin. Suppressive effects of sialic acid in the expression of biological activity of human erythropoietin in vitro. Eur. J. Biochem. 194:457‐462.
   Imperiali, B. and Rickert, K.W. 1995. Conformational implications of asparagine‐linked glycosylation. Proc. Natl. Acad. Sci. U.S.A. 92:97‐101.
   Inglese, J., Glickman, J.F., Lorenz, W., Caron, M.G., and Lefkowitz, R.J. 1992. Isoprenylation of a protein kinase. Requirement of farnesylation/α‐carboxyl methylation for full enzymatic activity of rhodopsin kinase. J. Biol. Chem. 267:1422‐1425.
   Itri, L.M. 1987. Report of Roche recombinant human interleukin‐2 clinical trials in cancer patients. J. Biol. Respir. Med. 6:220‐221.
   James, D.C., Freedman, R.B., Hoare, M., Ogonah, O.W., Rooney, B.C., Larionov, O.A., Dobrovolsky, V.N., Lagutin, O.V., and Jenkins, N. 1995. N‐glycosylation of recombinant human interferon‐γ produced in different animal expression systems. Bio/Technology 13:592‐596.
   Jarvis, D.L. and Finn, E.E. 1996. Modifying the insect cell N‐glycosylation pathway with immediate early baculovirus expression vectors. Nature Biotechnol. 14:1288‐1292.
   Jarvis, D.L., Kawar, Z.S., and Hollister, J.R. 1998. Engineering N‐glycosylation pathways in the baculovirus‐insect cell system. Curr. Opin. Biotechnol. 9:528‐533.
   Jefferis, R. and Lund, J. 1997. Glycosylation of antibody molecules: Structural and functional significance. Chem. Immunol. 65:111‐128.
   Jenkins, N., Parekh, R.B., and James, D.C. 1996. Getting the glycosylation right: Implications for the biotechnology industry. Nature Biotechnol. 14:975‐981.
   Jespersen, A.M., Christensen, T., Klausen, N.K., Nielsen, F., and Sorensen, H.H. 1994. Characterization of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli. Eur. J. Biochem. 219:365‐373.
   Kainuma, M., Ishida, N., Yoko‐o, T., Yoshioka, S., Takeuchi, M., Kawakita, M., and Jigami, Y. 1999. Coexpression of α 1,2 galactosyltransferase and UDP‐galactose transporter efficiently galactosylates N‐ and O‐glycans in Saccharomyces cerevisiae. Glycobiology 9:133‐141.
   Kamps, M.P., Buss, J.E., and Sefton, B.M. 1985. Mutation of NH2‐terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc. Natl. Acad. Sci. U.S.A. 82:4625‐4628.
   Kang, H.A., Sohn, J.H., Choi, E.S., Chung, B.H., Yu, M.H., and Rhee, S.K. 1998. Glycosylation of human alpha 1‐antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts. Yeast 14:371‐381.
   Kaplan, S.L., Underwood, L.E., August, G.P., Bell, J.J., Blethen, S.L., Blizzard, R.M., Brown, D.R., Foley, T.P., Hintz, R.L., Hopwood, N.J., et al. 1986. Clinical studies with recombinant‐DNA‐derived methionyl human growth hormone in growth hormone–deficient children. Lancet 1:697‐700.
   Katoh, S., Sezai, Y., Yamaguchi, T., Katoh, Y., Yagi, H., and Hohara, D. 1999. Refolding of enzymes in a fed‐batch operation. Proc. Biochem. 35:297‐300.
   Katre, N.V. 1990. Immunogenicity of recombinant IL‐2 modified by covalent attachment of polyethylene glycol. J. Immunol. 144:209‐213.
   Kaufman, R.J. 1998. Posttranslational modifications required for coagulation factor secretion and function. Thromb. Haemost. 79:1068‐1079.
   Kaufman, R.J., Wasley, L.C., Furie, B.C., Furie, B., and Schoemaker, C. 1986. Expression, purification and characterization of recombinant γ‐carboxylated factor IX synthesised in Chinese hamster ovary cells. J. Biol. Chem. 261:9622‐9628.
   Kern, G., Schulke, N., Schmid, F.X., and Jaenicke, R. 1992. Stability, quaternary structure, and folding of internal, external, and core‐glycosylated invertase from yeast. Protein Sci. 1:120‐131.
   Kitchin, K. and Flickinger, M.C. 1995. Alteration of hybridoma viability and antibody secretion in transfectomas with inducible overexpression of protein disulfide isomerase. Biotechnol. Prog. 11:565‐574.
   Kleid, D.G. 1983. Using genetically engineered bacteria for vaccine production. Ann. N.Y. Acad. Sci. 413:23‐30.
   Knauf, M.J., Bell, D.P., Hirtzer, P., Luo, Z.P., Young, J.D., and Katre, N.V. 1988. Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin‐2 chemically modified with water‐soluble polymers. J. Biol. Chem. 263:15064‐15070.
   Kornfield, R. and Kornfield, S. 1985. Assembly of asparagine‐linked oligosaccharides. Annu. Rev. Biochem. 54:631‐664.
   Krishna, R.G. and Wold, F. 1998. Posttranslational modifications. In Proteins: Analysis and Design (R.H. Angeletti, ed.) pp. 121‐206. Academic Press, San Diego.
   Kubota, H., Hyner, G., and Willison, K. 1995. The chaperonin containing t‐complex polypeptide 1 (TCP‐1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur. J. Biochem. 230:3‐16.
   Kudlicki, W., Odom, O.W., Kramer, G., Hardesty, B., Merrill, G.A., and Horowitz, P.M. 1995. The importance of the N‐terminal segment for DnaJ‐mediated folding of rhodanese while bound to ribosomes as peptidyl‐tRNA. J. Biol. Chem. 270:10650‐10657.
   Kulakosky, P.C., Hughes, P.R., and Wood, H.A. 1998. N‐Linked glycosylation of a baculovirus‐expressed recombinant glycoprotein in insect larvae and tissue culture cells. Glycobiology 8:741‐745.
   Kuo, M.H. and Allis, C.D. 1998. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20:615‐626.
   Laidler, P. and Litynska, A. 1997. Tumor cell N‐glycans in metastasis. Acta Biochim. Pol. 44:343‐357.
   La Vallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., and McKoy, J.M. 1993. A thioredoxin gene that circumvents inclusion body formaton in the E. coli cytoplasm. Biotechnology 11:187‐193.
   Lehle, L., Eiden, A., Lehnert, K., Haselbeck, A., and Kopetzki, E. 1995. Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N‐glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation. FEBS Lett. 370:41‐45.
   Lewis, S.A., Tian, G., Vainberg, I.E., and Cowan, N.J. 1996. Chaperonin‐mediated folding of actin and tubulin. J. Cell Biol. 132:1‐4.
   Lorimer, G.H. 1996. A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo. FASEB J. 10:5‐9.
   Mackay, V.L., Yip, C., Welch, S., Gilbert, T., Seidel, P., Grant, F., and O'Hara, P. 1990. Glycosylation and export of heterologous proteins expressed in yeast. In Recombinant Systems in Protein Expression (K.K. Alitalo, M‐L. Huhtala, J. Knowles, and A. Vaheri, eds.) pp. 25‐36. Elsevier Science Publishing, New York.
   Marino, M.H. 1989. Expression systems for heterologous protein production. Biopharm‐manuf. 2:18‐29, 32‐33.
   Marston, F.A.O. 1986. The purification of eukaryotic proteins synthesized in Escherichia coli. Biochem. J. 240:1‐12.
   Martinet, W., Saelens, X., Deroo, T., Neirynck, S., Contreras, R., Min Jou, W., and Fiers, W. 1997. Protection of mice against a lethal influenza challenge by immunization with yeast‐derived recombinant influenza neuraminidase. Eur. J. Biochem. 247:332‐338.
   Marullo, S., Delavier‐Klutchko, C., Eshdat, Y., Strosberg, A.D., and Emorine, L. 1988. Human β2‐adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties. Proc. Natl. Acad. Sci. U.S.A. 85:7551‐7555.
   Miele, R.G., Nilsen, S.L., Brito, T., Bretthauer, R.K., and Castellino, F.J. 1997. Glycosylation properties of the Pichia pastoris–expressed recombinant kringle 2 domain of tissue‐type plasminogen activator. Biotechnol. Appl. Biochem. 25:151‐157.
   Minch, S.L., Kallio, P.T., and Bailey, J.E. 1995. Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with α(2,6)‐sialyltransferase contains NeuAcα(2,6)Galβ(1,4)GlcNAcR linkages. Biotechnol. Prog. 11:348‐351.
   Missiakas, D., Georgopoulos, C., and Raina, S. 1993. Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. Proc. Natl. Acad. Sci. U.S.A. 90:7084‐7088.
   Mitraki, A., Fane, B., Haase‐Pettingell, C., Sturtevant, J., and King, J. 1991. Global suppression of protein folding defects and inclusion body formation. Science 253:54‐58.
   Montesino, R., Garcia, R., Quintero, O., and Cremata, J.A. 1998. Variation in N‐linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Express. Purif. 14:197‐207.
   Moonen, P., Mermod, J.J., Ernst, J.F., Hirschi, M., and DeLamarter, J.F. 1987. Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony‐stimulating factor produced by yeast or animal cells. Proc. Natl. Acad. Sci. U.S.A. 84:4428‐4431.
   Moore, W.V. and Leppert, P. 1980. Role of aggregated human growth hormone (hGH) in development of antibodies to hGH. J. Clin. Endocrinol. Metab. 51:691‐697.
   Morris, A.E., Lee, C.‐C., Hodges, K., Aldrich, T.L., Krantz, C., Smidt, P.S., and Thomas, J.N. 1997. Expression augmenting sequence element (EASE) isolated from Chinese hamster ovary cells. In Animal Cell Technology (M.J.T. Carrondo, ed.) pp. 529‐534. Kluwer Academic Publishers, Boston.
   Nelson, R.J., Ziegelhoffer, T., Nicolet, C., Werner‐Washburne, M., and Craig, E.A. 1993. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell 71:97‐105.
   Netzer, W.J. and Hartl, F.U. 1998. Protein folding in the cytosol: Chaperonin‐dependent and ‐independent mechanisms. Trends Biochem. Sci. 23:68‐73.
   Niehrs, C., Beisswanger, R., and Huttner, W.B. 1994. Protein tyrosine sulfation, 1993—an update. Chem. Biol. Interact. 92:257‐271.
   Nishihara, S., Narimatsu, M., Iwasaki, H., Yazawa, S., Akamatsu, S., Ando, T., Seno, T., and Nirimatsu, I. 1994. Molecular genetic analysis of the human Lewis histo‐blood group system. J. Biol. Chem. 269:29271‐29278.
   Ogonah, O.W., Freedman, R.B., Jenkins, N., Patel, K., and Rooney, B. 1996. Isolation and characterization of an insect cell line able to perform complex N‐linked glycosylation on recombinant proteins. Bio/Technology 14:197‐202.
   Oh‐eda, M., Hasegawa, M., Hattori, K., Kuboniwa, H., Kojima, T., Orita, T., Tomonou, K., Yamazaki, T., and Ochi, N. 1990. O‐linked sugar chain of human granulocyte colony‐stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity. J. Biol. Chem. 265:11432‐11435.
   Omary, M.B., Ku, N.O., Liao, J., and Price, D. 1998. Keratin modifications and solubility properties in epithelial cells and in vitro. Subcell. Biochem. 31:105‐140.
   O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1994. Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York.
   Quirk, A.V., Geisow, M.J., Woodrow, J.R., Burton, S.J., Wood, P.C., Sutton, A.D., Johnson, R.A., and Dodsworth, N. 1989. Production of recombinant human serum albumin from Saccharomyces cerevisiae. Biotechnol. Appl. Biochem. 11:273‐287.
   Ramer, S.E., Winkler, D.G., Carrera, A., Roberts, T.M., and Walsh, C.T. 1991. Purification and initial characterization of the lymphoid‐cell protein tyrosine kinase p56lck from a baculovirus expression system. Proc. Natl. Acad. Sci. U.S.A. 88:6254‐6258.
   Rhodes, C.J., Brennan, S.O., and Hutton, J.C. 1989. Proalbumin to albumin conversion by a proinsulin processing endopeptidase of insulin secretory granules. J. Biol. Chem. 264:14240‐14246.
   Riquelme, P.T., Buzzio, L.O., and Koide, S.S. 1979. ADP‐Ribosylation of rat liver lysine‐rich histone in vitro. J. Biol. Chem. 254:3018‐3028.
   Rudd, P.M., Joao, H.C., Coghill, E., Fiten, P., Saunders, M.R., Opdenakker, G., and Dwek, R.A. 1994. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 33:17‐22.
   Ruldolph, R. and Lilie, H. 1996. In vitro folding of inclusion body proteins. FASEB J. 10:49‐56.
   Schein, C.H. 1989. Production of soluble recombinant proteins in bacteria. Bio/Technology 7:1141‐1149.
   Schertler, G.F.X. 1992. Overproduction of membrane proteins. Curr. Opin. Struct. Biol. 2:534‐544.
   Schroder, M. and Friedl, P. 1997. Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein. Biotechnol. Bioeng. 53:547‐559.
   Schultz, L.D., Tanner, J., Hofmann, K.J., Emini, E.A., Condra, J.H., Jones, R.E., Kieff, E., and Ellis, R.W. 1987. Expression and secretion in yeast of a 400‐kDa envelope glycoprotein derived from Epstein‐Barr virus. Gene 54:113‐123.
   Sears, P. and Wang, C.‐H. 1998. Enzyme action in glycoprotein synthesis. Cell. Mol. Life Sci. 54:223‐252.
   Shusta, E.V., Raines, R.T., Pluckthun, A., and Wittrup, K.D. 1998. Increasing the secretory capacity of Saccharomyces cerevisiae for production of single‐chain antibody fragments. Nature Biotechnol. 16:773‐777.
   Sicheri, F., Moarefi, I., and Kuriyan, J. 1997. Crystal structure of the Src family tyrosine kinase Hck. Nature 385:602‐609.
   Stanley, P. 1992. Glycosylation engineering. Glycobiology 2:99‐107.
   Stanley, P. and Ioffe, E. 1995. Glycosyltransferase mutants: Key to new insights in glycobiology. FASEB J. 9:1436‐1444.
   Suenaga, M., Ohmae, H., Tsuji, S., Tanaka, Y., Koyama, N., and Nishimura, O. 1996. Epsilon‐N‐acetylation in the production of recombinant human basic fibroblast growth factor mutein. Prep. Biochem. Biotechnol. 26:59‐70.
   Sugino, Y., Tsunasawa, S., Yutani, K., Ogasahara, K., and Suzuki, M. 1980. Amino‐terminally formylated tryptophan synthase alpha‐subunit produced by the trp operon cloned in a plasmid vector. J. Biochem. 87:351‐354.
   Suster, J.R. 1989. Regulated transcriptional systems for the production of proteins in yeast: Regulation by carbon source. In Yeast Genetic Engineering (P.J. Barr, A.J. Brake, and P. Valenzuela, eds.) pp. 83‐108. Butterworths, London.
   Takeuchi, Y., Porter, C.D., Strahan, K.M., Preece, A.F., Gustafsson, K., Cosset, F.L., Weiss, R.A., and Collins, M.K. 1996. Sensitization of cells and retroviruses to human serum by α1,3 galactosyltransferase. Nature 379:85‐88.
   Tanigawara, Y., Hori, R., Okumura, K., Tsuji, J., Shimizu, N., Noma, S., Suzuki, J., Livingston, D.J., Richards, S.M., Keyes, L.D., et al. 1990. Pharmacokinetics in chimpanzees of recombinant human tissue‐type plasminogen activator produced in mouse C127 and Chinese hamster ovary cells. Chem. Pharm. Bull. 38:517‐522.
   Taylor, G., Hoare, M., Gray, D.R., and Marston, F.A.O. 1986. Size and density of protein inclusion bodies. Bio/Technology 4:553‐557.
   Thim, L., Bjoern, S., Christensen, M., Nicolaisen, E.M., Lund‐Hansen, T., Pedersen, A.H., and Hedner, U. 1988. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 27:7785‐7793.
   Thomas, G., Thorne, B.A., Thomas, L., Allen, R.G., Hroby, D.E., Fuller, R., and Thorner, J. 1988. Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science 241:226‐230.
   Thotakura, N.R. and Blithe, D.L. 1995. Glycoprotein hormones: Glycobiology of gonadotrophins, thyrotrophin and free alpha subunit. Glycobiology 5:3‐10.
   Travis, J.U., Owen, M., George, P., Carrell, R., Rosenberg, S., Hallewell, R.A., and Barr, P.J. 1985. Isolation and properties of recombinant DNA–produced variants of human α‐1 proteinase inhibitor. J. Biol. Chem. 260:4384‐4390.
   Trombetta, E.S. and Helenius, A. 1998. Lectins as chaperones in glycoprotein folding. Curr. Opin. Struct. Biol. 8:587‐592.
   Tsunasawa, S., Stewart, J.W., and Sherman, F. 1985. Amino‐terminal processing of mutant forms of yeast iso‐1‐cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase. J. Biol. Chem. 260:5382‐5391.
   Umana, P., Jean‐Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody‐dependent cellular cytotoxic activity. Nature Biotechnol. 17:176‐180.
   Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G., and Hall, B.D. 1982. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 298:347‐350.
   Van den Steen, P., Rudd, P.M., Dwek, R.A., and Opdenakker, G. 1998. Concepts and principles of O‐linked glycosylation. Crit. Rev. Biochem. Mol. Biol. 33:151‐208.
   van Die, I., van Tetering, A., Bakker, H., van den Eijnden, D.H., and Joziasse, D.H. 1996. Glycosylation in lepidopteran insect cells: Identification of a β 1 ⇒ 4‐N‐acetylgalactosaminyltransferase involved in the synthesis of complex‐type oligosaccharide chains. Glycobiology 6:157‐164.
   van Poelje, P.D. and Snell, E.E. 1990. Pyruvoyl‐dependent enzymes. Annu. Rev. Biochem. 59:29‐59.
   Varki, A. 1993. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 3:97‐130.
   Velardo, M.A., Bretthauer, R.K., Boutaud, A., Reinhold, B., Reinhold, V.N., and Castellino, F.J. 1993. The presence of UDP‐N‐acetylglucosamine: α‐3‐D‐mannoside β 1,2‐N‐acetylglucosaminyltransferase I activity in Spodoptera frugiperda cells (IPLB‐SF‐21AE) and its enhancement as a result of baculovirus infection. J. Biol. Chem. 268:17902‐17907.
   Wagenbach, M., O'Rourke, K., Vitez, L., Wieczorek, A., Hoffman, S., Durfee, S., Tedesco, J., and Stetler, G. 1991. Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae. Bio/Technology 9:57‐61.
   Wagner, R., Geyer, H., Geyer, R., and Klenk, H.D. 1996a. N‐Acetyl‐β‐glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector. J. Virol. 70:4103‐4109.
   Wagner, R., Liedtke, S., Kretzschmar, E., Geyer, H., Geyer, R., and Klenk, H.D. 1996b. Elongation of the N‐glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human β 1,2‐N‐acetylglucosaminyltransferase I. Glycobiology 6:165‐175.
   Weir, M.P. and Sparks, J. 1987. Purification and renaturation of recombinant human interleukin‐2. Biochem. J. 245:85‐91.
   Wilkinson, D.L. and Harrison, R.G. 1991. Predicting the solubility of recombinant proteins in Escherichia coli. Bio/Technology 9:443‐448.
   Wingfield, P., Pain, R.H., and Craig, S. 1987. Tumour necrosis factor is a compact trimer. FEBS Lett. 211:179‐184.
   Wingfield, P.T., Stahl, S.J., Williams, R.W., and Steven, A.C. 1995. Hepatitis core antigen produced in Escherichia coli: Subunit composition, conformational analysis, and in vitro capsid assembly. Biochemistry 34:4919‐4932.
   Wittwer, A.J. and Howard, S.C. 1990. Glycosylation at Asn‐184 inhibits the conversion of single‐chain to two‐chain tissue‐type plasminogen activator by plasmin. Biochemistry 29:4175‐4180.
   Wittwer, A.J., Howard, S.C., Carr, L.S., Harakas, N.K., Feder, J., Parekh, R.B., Rudd, P.M., Dwek, R.A., and Rademacher, T.W. 1989. Effects of N‐glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast–derived tissue plasminogen activator. Biochemistry 28:7662‐7669.
   Wood, C.R., Boss, M.A., Kenten, J.H., Calvert, J.E., Roberts, N.A., and Emtage, J.S. 1985. The synthesis and in vivo assembly of functional antibodies in yeast. Nature 314:445‐449.
   Wright, A. and Morrison, S.L. 1997. Effect of glycosylation on antibody function: Implications for genetic engineering. Trends Biotechnol. 15:26‐32.
   Wurm, F. and Bernard, A. 1999. Large‐scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10:156‐159.
   Xu, W., Harrison, S.E., and Eck, M.J. 1997. Three‐dimensional structure of the tyrosine kinase c‐Src. Nature 385:595‐602.
   Yan, S.C., Razzano, P., Chao, Y.B., Walls, J.D., Berg, D.T., McClure, D.B., and Grinnell, B.W. 1990. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Biotechnology 8:655‐661.
   Yarranton, G.T. 1992. High‐level expression in Escherichia coli. In Transgenesis: Applications of Gene Transfer (J.A.H. Murray, ed.) pp. 1‐29. John Wiley & Sons, New York.
   Youakim, A. and Shur, B.D. 1994. Alteration of oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases. Ann. N.Y. Acad. Sci. 745:331‐335.
   Zanghi, J.A., Mendoza, T.P., Schmelzer, A.E., Knop, R.H., and Miller, W.M. 1998. Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia. Biotechnol. Prog. 14:834‐844.
   Zhang, X., Lok, S.H., and Kon, O.L. 1998. Stable expression of human α‐2,6‐sialyltransferase in Chinese hamster ovary cells: Functional consequences for human erythropoietin expression and bioactivity. Biochim. Biophys. Acta 1425:441‐452.
   Zimmerman, S.B. and Trach, S.O. 1991. Estimaton of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 222:599‐620.
Internet Resources
   http://www.fda.gov
  U.S. Food and Drug Administration Web site. Provides governmental information on replacing animal‐derived products with non‐animal products when using expressed protein for human clinical applications.
   http://www.eudra.org
  European site providing similar information.
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