Molecular and Cellular Biochemistry

, Volume 307, Issue 1–2, pp 249–264 | Cite as

Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies

  • Sudhir Sahdev
  • Sunil K. Khattar
  • Kulvinder Singh Saini


Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein’s over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.


Post-translational modifications Disulfide bond Phosphorylation Glycosylation Inclusion body refolding Size exclusion chromatography Hydrophobic interactions 



This work was funded by Ranbaxy Research Laboratories, India. We thankfully acknowledge our colleagues within the New Drug Discovery Research Group, Department of Biotechnology Team at Ranbaxy, particularly Dr. R.S. Bora, Dr. R. Kant, Mr. Roop Trivedi for their assistance during review compilation and Mr. Prabhakar Tiwari, Ms. Shalini Saini for generating the unpublished data.


  1. 1.
    Nakamura Y, Gojobori T, Ikemura T (2000) Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 29:292Google Scholar
  2. 2.
    Sharp PM, Devine KM (1989) Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ‘prefer’ optimal codons. Nucleic Acids Res 17:5029–5039PubMedGoogle Scholar
  3. 3.
    Rosenberg AH, Goldman E, Dunn JJ et al (1993) Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bacteriol 175:716–722PubMedGoogle Scholar
  4. 4.
    Brinkmann U, Mattes RE, Buckel P (1989) High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the DnaY gene product. Gene 85:109–114PubMedGoogle Scholar
  5. 5.
    Cinquin O, Christopherson RI, Menz RI (2001) A hybrid plasmid for expression of toxic malarial proteins in Escherichia coli. Mol Biochem Parasitol 117:245–247PubMedGoogle Scholar
  6. 6.
    Yu ZB, Jin JP (2007) Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression. Arch Biochem Biophys 461:138–145PubMedGoogle Scholar
  7. 7.
    Trundova M, Celer V (2007) Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells. Virus Genes 34:199–204PubMedGoogle Scholar
  8. 8.
    Kirienko NV, Lepikhov KA, Zheleznaya LA et al (2004) Significance of codon usage and irregularities of rare codon distribution in genes for expression of BspLU11III methyltransferases. Biochemistry (Mosc) 69:527–535Google Scholar
  9. 9.
    Sorensen HP, Sperling-Petersen HU, Mortensen KK (2003) Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck. J Chromatogr B Analyt Technol Biomed Life Sci 786:207–214PubMedGoogle Scholar
  10. 10.
    Neubauer A, Soini J, Bollok M et al (2007) Fermentation process for tetrameric human collagen prolyl 4-hydroxylase in Escherichia coli: improvement by gene optimisation of the PDI/beta subunit and repeated addition of the inducer anhydrotetracycline. J Biotechnol 128:308–321PubMedGoogle Scholar
  11. 11.
    Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51(1):1–10. ReviewPubMedGoogle Scholar
  12. 12.
    Andersen CL, Matthey-Dupraz A, Missiakas D et al (1997) A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins. Mol Microbiol 26:121–132PubMedGoogle Scholar
  13. 13.
    Liu X, Wang CC (2001) Disulfide-dependent folding and export of Escherichia coli DsbC. J Biol Chem 276:1146–1151PubMedGoogle Scholar
  14. 14.
    Kurokawa Y, Yanagi H, Yura T (2000) Overexpression of protein disulfide isomerase DsbC stabilizes multiple-disulfide-bonded recombinant protein produced and transported to the periplasm in Escherichia coli. Appl Environ Microbiol 66:3960–3965PubMedGoogle Scholar
  15. 15.
    Kurokawa Y, Yanagi H, Yura T (2001) Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli. J Biol Chem 276:14393–14399PubMedGoogle Scholar
  16. 16.
    Levy R, Weiss R, Chen G et al (2001) Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones. Protein Expr Purif 23:338–347PubMedGoogle Scholar
  17. 17.
    Jurado P, de Lorenzo V, Fernandez LA (2006) Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin. J Mol Biol 357:49–61PubMedGoogle Scholar
  18. 18.
    Bessette PH, Aslund F, Beckwith J et al (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA 96:13703–13708PubMedGoogle Scholar
  19. 19.
    Lilie H, McLaughlin S, Freedman R et al (1994) Influence of protein disulfide isomerase (PDI) on antibody folding in vitro. J Biol Chem 269:14290–14296PubMedGoogle Scholar
  20. 20.
    Klappa P, Hawkins HC, Freedman RB (1997) Interactions between protein disulphide isomerase and peptides. Eur J Biochem 248:37–42PubMedGoogle Scholar
  21. 21.
    Winter J, Klappa P, Freedman RB et al (2002) Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin. J Biol Chem 277:310–317PubMedGoogle Scholar
  22. 22.
    Chew CC, Magallon T, Martinat N et al (1995) The relative protein disulphide isomerase (PDI) activities of gonadotrophins, thioredoxin and PDI. Biochem Soc Trans 23:394SPubMedGoogle Scholar
  23. 23.
    Masuda K, Kamimura T, Kanesaki M et al (1996) Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli. Protein Eng 9:101–106PubMedGoogle Scholar
  24. 24.
    Mukhopadhyay A (2000) Reversible protection of disulfide bonds followed by oxidative folding render recombinant hCGbeta highly immunogenic. Vaccine 18:1802–1810PubMedGoogle Scholar
  25. 25.
    Yue BG, Ajuh P, Akusjarvi G et al (2000) Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli. Nucleic Acids Res 28:E14PubMedGoogle Scholar
  26. 26.
    Gosse ME, Padmanabhan A, Fleischmann RD et al (1993) Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits. Proc Natl Acad Sci USA 90:8159–8163PubMedGoogle Scholar
  27. 27.
    Mijakovic I, Petranovic D, Macek B et al (2006) Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine. Nucleic Acids Res 34:1588–1596PubMedGoogle Scholar
  28. 28.
    Mironova R, Niwa T, Handzhiyski Y et al (2005) Evidence for non-enzymatic glycosylation of Escherichia coli chromosomal DNA. Mol Microbiol 55:1801–1811PubMedGoogle Scholar
  29. 29.
    Zhang Z, Gildersleeve J, Yang YY et al (2004) A new strategy for the synthesis of glycoproteins. Science 303:371–373PubMedGoogle Scholar
  30. 30.
    Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J Mol Biol 146:1–21PubMedGoogle Scholar
  31. 31.
    Dong H, Nilsson L, Kurland CG (1996) Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J Mol Biol 260:649–663PubMedGoogle Scholar
  32. 32.
    Kane JF (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6:494–500PubMedGoogle Scholar
  33. 33.
    Kurland C, Gallant J (1996) Errors of heterologous protein expression. Curr Opin Biotechnol 7:489–493PubMedGoogle Scholar
  34. 34.
    Goldman E, Rosenberg AH, Zubay G et al (1995) Consecutive low-usage leucine codons block translation only when near the 5′ end of a message in Escherichia coli. J Mol Biol 245:467–473PubMedGoogle Scholar
  35. 35.
    Ahmed AK, Schaffer SW, Wetlaufer DB (1975) Non-enzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxido-reduction buffers. J Biol Chem 250:8477–8482PubMedGoogle Scholar
  36. 36.
    Wetlaufer DB, Branca PA, Chen GX (1987) The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential. Protein Eng 1:141–146PubMedGoogle Scholar
  37. 37.
    Gough JD, Gargano JM, Donofrio AE et al (2003) Aromatic thiol pKa effects on the folding rate of a disulfide containing protein. Biochemistry 42:11787–11797PubMedGoogle Scholar
  38. 38.
    DeCollo TV, Lees WJ (2001) Effects of aromatic thiols on thiol-disulfide interchange reactions that occur during protein folding. J Org Chem 66:4244–4249PubMedGoogle Scholar
  39. 39.
    Garnak M, Reeves HC (1979) Phosphorylation of Isocitrate dehydrogenase of Escherichia coli. Science 203:1111–1112PubMedGoogle Scholar
  40. 40.
    Yang L, Liu ZR (2004) Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues. Protein Expr Purif 35:327–333PubMedGoogle Scholar
  41. 41.
    El-Battari A, Prorok M, Angata K et al (2003) Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation. Glycobiology 13:941–953PubMedGoogle Scholar
  42. 42.
    Mironova R, Niwa T, Hayashi H et al (2001) Evidence for non-enzymatic glycosylation in Escherichia coli. Mol Microbiol 39:1061–1068PubMedGoogle Scholar
  43. 43.
    Jonasson P, Liljeqvist S, Nygren PA et al (2002) Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem 35:91–105PubMedGoogle Scholar
  44. 44.
    Ewis HE, Lu CD (2005) Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins. FEMS Microbiol Lett 253:295–301PubMedGoogle Scholar
  45. 45.
    Ignatova Z, Mahsunah A, Georgieva M et al (2003) Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains. Appl Environ Microbiol 69:1237–1245PubMedGoogle Scholar
  46. 46.
    Malik A, Rudolph R, Sohling B (2006) A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm. Protein Expr Purif 47:662–671PubMedGoogle Scholar
  47. 47.
    Oelschlaeger P, Lange S, Schmitt J et al (2003) Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression. Appl Microbiol Biotechnol 61:123–132PubMedGoogle Scholar
  48. 48.
    Griswold KE, Mahmood NA, Iverson BL et al (2003) Effects of codon usage versus putative 5′-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm. Protein Expr Purif 27:134–142PubMedGoogle Scholar
  49. 49.
    Adler J, Bibi E (2002) Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain. J Bacteriol 184:3313–3320PubMedGoogle Scholar
  50. 50.
    Obukowicz MG, Turner MA, Wong EY et al (1988) Secretion and export of IGF-1 in Escherichia coli strain JM101. Mol Gen Genet 215:19–25PubMedGoogle Scholar
  51. 51.
    Arie JP, Miot M, Sassoon N et al (2006) Formation of active inclusion bodies in the periplasm of Escherichia coli. Mol Microbiol 62:427–437PubMedGoogle Scholar
  52. 52.
    Jappelli R, Brenner S (1998) Changes in the periplasmic linker and in the expression level affect the activity of ToxR and lambda-ToxR fusion proteins in Escherichia coli. FEBS Lett 423:371–375PubMedGoogle Scholar
  53. 53.
    Tinker JK, Erbe JL, Holmes RK (2005) Characterization of fluorescent chimeras of cholera toxin and Escherichia coli heat-labile enterotoxins produced by use of the twin arginine translocation system. Infect Immun 73:3627–3635PubMedGoogle Scholar
  54. 54.
    Froderberg L, Houben EN, Baars L et al (2004) Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway. J Biol Chem 279:31026–31032PubMedGoogle Scholar
  55. 55.
    Rietsch A, Belin D, Martin N et al (1996) An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc Natl Acad Sci USA 93:13048–13053PubMedGoogle Scholar
  56. 56.
    Ostermeier M, De Sutter K, Georgiou G (1996) Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds. J Biol Chem 271:10616–10622PubMedGoogle Scholar
  57. 57.
    Chen R, Henning UA (1996) Periplasmic protein (SKP) of Escherichia coli selectively binds a class of outer membrane proteins. Mol Microbiol 19:1287–1294PubMedGoogle Scholar
  58. 58.
    Schafer U, Beck K, Muller M (1999) SKP, a Molecular chaperone of gram negative bacteria is required for formation of soluble periplasmic intermediates of outer membrane proteins. J Biol Chem 274:24567–24574PubMedGoogle Scholar
  59. 59.
    Missiakas D, Betton JM, Raina S (1996) New components of protein folding in extra-cytoplasmic components of Escherichia coli SurA, FkpA and Skp/OmpH. Mol Microbiol 21:871–884PubMedGoogle Scholar
  60. 60.
    Arie JP, Sasson N, Betton JM (2001) Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol Microbiol 39:199–210PubMedGoogle Scholar
  61. 61.
    Bothmann H, Pluckthun A (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J Biol Chem 275:17100–17105PubMedGoogle Scholar
  62. 62.
    Dartigalongue C, Raina S (1998) A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli. EMBO J 17:3968–3980PubMedGoogle Scholar
  63. 63.
    Segatori L, Murphy L, Arredondo S et al (2006) Conserved role of the linker alpha-helix of the bacterial disulfide isomerase DsbC in the avoidance of misoxidation by DsbB. J Biol Chem 281:4911–4919PubMedGoogle Scholar
  64. 64.
    Segatori L, Paukstelis PJ, Gilbert HF et al (2004) Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways. Proc Natl Acad Sci USA 101:10018–10023PubMedGoogle Scholar
  65. 65.
    Debarbieux L, Beckwith J (1998) The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc Natl Acad Sci USA 95:10751–10756PubMedGoogle Scholar
  66. 66.
    Powers T, Walter P (1997) Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor. EMBO J 16:4880–4886PubMedGoogle Scholar
  67. 67.
    Powers T, Walter P (1996) The nascent polypeptide-associated complex modulates interactions between the signal recognition particle and the ribosome. Curr Biol 6:331–338PubMedGoogle Scholar
  68. 68.
    Bacher G, Lutcke H, Jungnickel B et al (1996) Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting. Nature 381:248–251PubMedGoogle Scholar
  69. 69.
    Spanggord RJ, Siu F, Ke A et al (2005) RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle. Nat Struct Mol Biol 12:1116–1122PubMedGoogle Scholar
  70. 70.
    Miller JD, Wilhelm H, Gierasch L A et al (1993) GTP binding and hydrolysis by the signal recognition particle during initiation of protein translocation. Nature 366:351–354PubMedGoogle Scholar
  71. 71.
    Humphreys DP, Sehdev M, Chapman AP et al (2000) High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5′ end of the coding sequence. Protein Expr Purif 20:252–264PubMedGoogle Scholar
  72. 72.
    Rockenbach SK, Dupuis MJ, Pitts TW et al (1991) Secretion of active truncated CD4 into Escherichia coli periplasm. Appl Microbiol Biotechnol 35:32–37PubMedGoogle Scholar
  73. 73.
    Bolla JM, Lazdunski C, Inouye M et al (1987) Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli. FEBS Lett 224:213–218PubMedGoogle Scholar
  74. 74.
    Neumann-Haefelin C, Schafer U, Muller M et al (2000) SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein. EMBO J 19:6419–6426PubMedGoogle Scholar
  75. 75.
    Eichler J, Wickner W (1998) The SecA subunit of Escherichia coli preprotein translocase is exposed to the periplasm. J Bacteriol 180:5776–5779PubMedGoogle Scholar
  76. 76.
    Kim J, Lee Y, Kim C et al (1992) Involvement of SecB, a chaperone, in the export of ribose binding protein. J Bacteriol 174:5219–5227PubMedGoogle Scholar
  77. 77.
    Hunt I (2005) From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expr Purif 40(1):1–22. ReviewPubMedGoogle Scholar
  78. 78.
    Donovan RS, Robinson CW, Glick BR (2000) Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter. Can J Microbiol 46:532–541PubMedGoogle Scholar
  79. 79.
    Vera A, Gonzalez-Montalban N, Aris A et al (2007) The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 96:1101–1106PubMedGoogle Scholar
  80. 80.
    Chesshyre JA, Hipkiss AR (1989) Low temperatures stabilize interferon a-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Appl Microbiol Biotechnol 31:158–162Google Scholar
  81. 81.
    Niiranen L, Espelid S, Karlsen CR et al (2007) Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. Protein Expr Purif 52:210–218PubMedGoogle Scholar
  82. 82.
    Ferrer M, Chernikova TN, Yakimov MM (2003) Chaperons govern growth of Escherichia coli at low temperature. Nat Biotechnol 21:1266–1267PubMedGoogle Scholar
  83. 83.
    Hunke S, Betton JM (2003) Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli. Mol Microbiol 50:1579–1589PubMedGoogle Scholar
  84. 84.
    Spiess C, Beil A, Ehrmann M (1999) A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97:339–347PubMedGoogle Scholar
  85. 85.
    Vasina JA, Baneyx F (1997) Expression of aggregation prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr Purif 9:211–218PubMedGoogle Scholar
  86. 86.
    Phadtare S, Severinov K (2005) Extended -10 motif is critical for activity of the cspA promoter but does not contribute to low-temperature transcription. J Bacteriol 187:6584–6589PubMedGoogle Scholar
  87. 87.
    Fang L, Xia B, Inouye M (1999) Transcription of cspA, the gene for the major cold-shock protein of Escherichia coli, is negatively regulated at 37 degrees C by the 5′-untranslated region of its mRNA. FEMS Microbiol Lett 176:39–43PubMedGoogle Scholar
  88. 88.
    Shaw MK, Ingraham JL (1967) Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth. J Bacteriol 94:157–164PubMedGoogle Scholar
  89. 89.
    Schmidt M, Viaplana E, Hoffmann F (1999) Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway. Biotechnol Bioeng 66:61–67PubMedGoogle Scholar
  90. 90.
    Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219:37–44PubMedGoogle Scholar
  91. 91.
    Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59PubMedGoogle Scholar
  92. 92.
    Picaud S, Olsson ME, Brodelius PE (2007) Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli. Protein Expr Purif 51:71–79PubMedGoogle Scholar
  93. 93.
    Saejung W, Puttikhunt C, Prommool T et al (2006) Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in Escherichia coli trxB and gor double mutant. J Biosci Bioeng 102:333–339PubMedGoogle Scholar
  94. 94.
    Boccazzi P, Zanzotto A et al (2005) Gene expression analysis of Escherichia coli grown in miniaturized bioreactor platforms for high-throughput analysis of growth and genomic data. Appl Microbiol Biotechnol 68:518–532PubMedGoogle Scholar
  95. 95.
    Losen M, Frolich B, Pohl M et al (2004) Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures. Biotechnol Prog 20:1062–1068PubMedGoogle Scholar
  96. 96.
    Broedel SE Jr, Papciak SM et al (2001) The selection of optimum media formulations for improved expression of recombinant proteins in E. coli, vol 2. Athena Environmental Sciences, IncGoogle Scholar
  97. 97.
    Bruser T, Yano T, Brune DC et al (2003) Membrane targeting of a folded and cofactor-containing protein. Eur J Biochem 270:1211–1221PubMedGoogle Scholar
  98. 98.
    Apiyo D, Wittung-Stafshede P (2002) Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin. Protein Sci 11:1129–1135PubMedGoogle Scholar
  99. 99.
    Van Dien SJ, Iwatani S, Usuda Y et al (2006) Theoretical analysis of amino acid-producing Escherichia coli using a stoichiometric model and multivariate linear regression. J Biosci Bioeng 102:34–40PubMedGoogle Scholar
  100. 100.
    Lee KM, Gilmore DF (2006) Statistical experimental design for bioprocess modeling and optimization analysis: repeated-measures method for dynamic biotechnology process. Appl Biochem Biotechnol 135:101–116PubMedGoogle Scholar
  101. 101.
    Khattar SK, Gulati P, Kundu PK et al (2007) Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli. Protein Pept Lett 14(8):756–760PubMedGoogle Scholar
  102. 102.
    Rathore AS, Sobacke SE, Kocot TJ et al (2003) Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells. J Pharm Biomed Anal 32:1199–1211PubMedGoogle Scholar
  103. 103.
    Deuerling E, Patzelt H, Vorderwulbecke S et al (2003) Trigger factor and DnaK possess overlapping substrate pools and binding specificities. Mol Microbiol 47:1317–1328PubMedGoogle Scholar
  104. 104.
    Nishihara K, Kanemori M, Yanagi H et al (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 66:884–889PubMedGoogle Scholar
  105. 105.
    Ehrnsperger M, Graber S, Gaestel M et al (1997) Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J 16:221–229PubMedGoogle Scholar
  106. 106.
    Veinger L, Diamant S, Buchner J et al (1998) The small heat-shock protein IbpB from Escherichia coli stabilize stress-denatured proteins for subsequent refolding by a multi-chaperone network. J Biol Chem 273:11032–11037PubMedGoogle Scholar
  107. 107.
    Hu X, O’Hara L, White S et al (2007) Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support. Protein Expr Purif 52:194–201PubMedGoogle Scholar
  108. 108.
    Kondo A, Kohda J, Endo Y et al (2000) Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins. J Biosci Bioeng 90:600–606PubMedGoogle Scholar
  109. 109.
    Xu Y, Hsieh MY, Narayanan N et al (2005) Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli. Biotechnol Prog 21:1357–1365PubMedGoogle Scholar
  110. 110.
    Kitagawa M, Miyakawa M, Matsumura Y et al (2002) Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. Eur J Biochem 269:2907–2917PubMedGoogle Scholar
  111. 111.
    Kuczynska-Wisnik D, Kedzierska S, Matuszewska E et al (2002) The Escherichia coli small heat-shock proteins IbpA and IbpB prevent the aggregation of endogenous proteins denatured in vivo during extreme heat shock. Microbiology 148:1757–1765PubMedGoogle Scholar
  112. 112.
    Zolkiewski M (1999) ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli. J Biol Chem 274:28083–28086PubMedGoogle Scholar
  113. 113.
    Thomas JG, Baneyx F (2000) ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. Mol Microbiol 36:1360–1370PubMedGoogle Scholar
  114. 114.
    Zavilgelsky GB, Kotova VY, Mazhul MM et al (2002) Role of Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA and ClpB) chaperones in refolding and increased thermal stability of bacterial luciferases in Escherichia coli cells. Biochemistry (Mosc) 67:986–992Google Scholar
  115. 115.
    Xu HM, Zhang GY, Ji XD et al (2005) Expression of soluble, biologically active recombinant human endostatin in Escherichia coli. Protein Expr Purif 41:252–258PubMedGoogle Scholar
  116. 116.
    Yoshimune K, Ninomiya Y, Wakayama M et al (2004) Molecular chaperones facilitate the soluble expression of N-acyl-D-amino acid amidohydrolases in Escherichia coli. J Ind Microbiol Biotechnol 31:421–426PubMedGoogle Scholar
  117. 117.
    Ghosh S, Rasheedi S, Rahim SS et al (2004) Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli. Biotechniques 37:418–423PubMedGoogle Scholar
  118. 118.
    Patra AK, Gahlay GK, Reddy BV et al (2000) Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli. Eur J Biochem 267:7075–7081PubMedGoogle Scholar
  119. 119.
    Patra AK, Mukhopadhyay R, Mukhija R et al (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 18:182–192PubMedGoogle Scholar
  120. 120.
    Kwon KS, Lee S, Yu MH (1995) Refolding of alpha 1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation. Biochim Biophys Acta 1247:179–184PubMedGoogle Scholar
  121. 121.
    Venkiteshwaran A, Heider P, Matosevic S et al (2007) Optimized removal of soluble host cell proteins for the recovery of met-Human growth hormone inclusion bodies from Escherichia coli cell lysate using crossflow microfiltration. Biotechnol Prog May 5 [Epub ahead of print]Google Scholar
  122. 122.
    Batas B, Schiraldi C, Chaudhuri JB (1999) Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography. J Biotechnol 68:149–158PubMedGoogle Scholar
  123. 123.
    Oneda H, Inouye K (1999) Refolding and recovery of recombinant human matrix metalloproteinase 7 (matrilysin) from inclusion bodies expressed by Escherichia coli. J Biochem (Tokyo) 126:905–911Google Scholar
  124. 124.
    Sunitha K, Chung BH, Jang KH et al (2000) Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli. Protein Expr Purif 18:388–393PubMedGoogle Scholar
  125. 125.
    Lopez-Vara MC, Gasset M, Pajares MA (2000) Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies. Protein Expr Purif 19:219–226PubMedGoogle Scholar
  126. 126.
    Guisez Y, Demolder J, Mertens N et al (1993) High-level expression, purification, and renaturation of recombinant murine interleukin-2 from Escherichia coli. Protein Expr Purif 4:240–246PubMedGoogle Scholar
  127. 127.
    Lim WK, Smith-Somerville HE, Hardman JK (1989) Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits. Appl Environ Microbiol 55:1106–1111PubMedGoogle Scholar
  128. 128.
    Rinas U, Hoffmann F, Betiku E et al (2007) Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. J Biotechnol 127:244–257PubMedGoogle Scholar
  129. 129.
    Lu H, Yu M, Sun Y et al (2007) Expression and purification of bioactive high-purity mouse monokine induced by IFN-gamma in Escherichia coli. Protein Expr Purif Apr 14 [Epub ahead of print]Google Scholar
  130. 130.
    Liu X, He Z, Zhou M et al (2007) Purification and characterization of recombinant extracellular domain of human HER2 from Escherichia coli. Protein Expr Purif 53:247–254PubMedGoogle Scholar
  131. 131.
    Li M, Huang D (2007) On-column refolding purification and characterization of recombinant human interferon-lambda1 produced in Escherichia coli. Protein Expr Purif 53:119–123PubMedGoogle Scholar
  132. 132.
    Bruser T (2007) The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production. Appl Microbiol Biotechnol [Epub ahead of print]Google Scholar
  133. 133.
    Takahashi S, Ogasawara H, Watanabe T et al (2006) Refolding and activation of human prorenin expressed in Escherichia coli: application of recombinant human renin for inhibitor screening. Biosci Biotechnol Biochem 70:2913–2918PubMedGoogle Scholar
  134. 134.
    Hagel P, Gerding JJT, Fieggen W et al (1971) Cyanate formation in solutions of urea I. Calculation of cyanate concentrations at different temperature and pH. Biochim Biophys Acta 243:366–373PubMedGoogle Scholar
  135. 135.
    Boyle DM, McKinnie RE, Joy WD et al (1999) Evaluation of refolding conditions for a recombinant human interleukin-3 variant (daniplestim). Biotechnol Appl Biochem 30:163–170PubMedGoogle Scholar
  136. 136.
    De Bernardez Clark E, Schwarz E, Rudolph R (1999) Inhibition of aggregation side reactions during in vitro protein folding. Methods Enzymol 309:217–236PubMedCrossRefGoogle Scholar
  137. 137.
    Kurucz I, Titus JA, Jost CR et al (1995) Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies. Mol Immunol 2:1443–1452Google Scholar
  138. 138.
    Tsumoto K, Umetsu M, Kumagai I et al (2003) Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. Biochem Biophys Res Commun 312:1383–1386PubMedGoogle Scholar
  139. 139.
    Goulding CW, Perry LJ (2003) Protein production in Escherichia coli for structural studies by X-ray crystallography. J Struct Biol 142:133–143. ReviewPubMedGoogle Scholar
  140. 140.
    Tokatlidis K, Dhurjati P, Millet J et al (1991) High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D. FEBS Lett 282:205–208PubMedGoogle Scholar
  141. 141.
    Puri NK, Crivelli E, Cardamone M et al (1992) Solubilization of growth hormone and other recombinant proteins from Escherichia coli by using a cationic surfactant. Biochem J 285:871–879PubMedGoogle Scholar
  142. 142.
    Khan RH, Appa Rao KBC, Eshwari ANS et al (1998) Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli. Biotechnol Prog 14:722–728PubMedGoogle Scholar
  143. 143.
    Hale JE, Butler JP, Gelfanova V et al (2004) A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis. Anal Biochem 333:174–181PubMedGoogle Scholar
  144. 144.
    Cindric M, Cepo T, Skrlin A et al (2006) Accelerated on-column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis. Rapid Commun Mass Spectrom 20:694–702PubMedGoogle Scholar
  145. 145.
    Righetti PG, Chiari M, Casale E et al (1989) Oxidation of alkaline immobiline buffers for isoelectric focusing in immobilized pH gradients. Appl Theor Electrophor 1:115–121PubMedGoogle Scholar
  146. 146.
    Danek BL, Robinson AS (2004) P22 tailspike trimer assembly is governed by interchain redox associations. Biochim Biophys Acta 1700:105–116PubMedGoogle Scholar
  147. 147.
    Lee SH, Carpenter JF, Chang BS et al (2006) Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure. Protein Sci 15:304–313PubMedGoogle Scholar
  148. 148.
    Tran-Moseman A, Schauer N, De Bernardez Clark E (1999) Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor. Protein Expr Purif 16:181–189PubMedGoogle Scholar
  149. 149.
    Rudolph R, Zettlmeissl G, Jaenicke R (1979) Reconstitution of lactic dehydrogenase. Non-covalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates. Biochemistry 18:5572–5575PubMedGoogle Scholar
  150. 150.
    Goldberg ME, Rudolph R, Jaenicke R (1991) A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. Biochemistry 30:2790–2797PubMedGoogle Scholar
  151. 151.
    De Bernardez Clark E, Hevehan D, Szela S et al (1998) Oxidative renaturation of hen egg-white lysozyme. Folding vs. aggregation. Biotechnol Prog 14:47–54PubMedGoogle Scholar
  152. 152.
    Vallejo LF, Rinas U (2004) Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration. Biotechnol Bioeng 85:601–609PubMedGoogle Scholar
  153. 153.
    Buchner J, Pastan I, Brinkmann U (1992) A method for increasing the yield of properly folded recombinant fusion proteins: Single chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem 205:263–270PubMedGoogle Scholar
  154. 154.
    Fischer B, Perry B, Sumner I et al (1992) A novel sequential procedure to enhance the renaturation of recombinant protein from Escherichia coli inclusion bodies. Protein Eng 5:593–596PubMedGoogle Scholar
  155. 155.
    Terashima M, Suzuki K, Katoh S (1996) Effective refolding of fully reduced lysozyme with a flow-type reactor. Process Biochem 31:341–345Google Scholar
  156. 156.
    Ho JGS, Middelberg APJ, Ramage P et al (2003) The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient. Protein Sci 12:708–716PubMedGoogle Scholar
  157. 157.
    Maeda Y, Koga H, Yamada H (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng 8:201–205PubMedGoogle Scholar
  158. 158.
    Varnerin JP, Smith T, Rosenblum CI et al (1998) Production of leptin in Escherichia coli: a comparison of methods. Protein Expr Purif 14:335–342PubMedGoogle Scholar
  159. 159.
    West SM, Chaudhuri JB, Howell JA (1998) Improved protein refolding using hollow-fibre membrane dialysis. Biotechnol Bioeng 57:590–599PubMedGoogle Scholar
  160. 160.
    Yoshii H, Furuta T, Yonehara T et al (2000) Refolding of denatured/reduced lysozyme at high concentration with diafiltration. Biosci Biotechnol Biochem 64:1159–1165PubMedGoogle Scholar
  161. 161.
    Gu Z, Weidenhaupt M, Ivanova N et al (2002) Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies. Protein Express Purif 25:174–179Google Scholar
  162. 162.
    Müller C, Rinas U (1999) Renaturation of heterodimeric platelet derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography. J Chromatogr A 855:203–213PubMedGoogle Scholar
  163. 163.
    Werner MH, Clore GM, Gronenborn AM et al (1994) Refolding proteins by gel filtration chromatography. FEBS Lett 345:125–130PubMedGoogle Scholar
  164. 164.
    Batas B, Chaudhuri JB (1996) Protein refolding at high concentration using size-exclusion chromatography. Biotechnol Bioeng 50:16–23PubMedGoogle Scholar
  165. 165.
    Batas B, Chaudhuri JB (1999) Considerations of sample application and elution during size-exclusion chromatography-based protein refolding. J Chromatogr A 864:229–236PubMedGoogle Scholar
  166. 166.
    Gu Z, Su Z, Janson J-C (2001) Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding. J Chromatogr A 918:311–318PubMedGoogle Scholar
  167. 167.
    Fahey EM, Chaudhuri JB (2000) Refolding of low molecular weight urokinase plasminogen activator by dilution and size exclusion chromatography-a comparative study. Sep Sci Technol 35:1743–1760Google Scholar
  168. 168.
    Li M, Poliakov A, Danielson UH et al (2003) Refolding of a recombinant full-length non-structural (NS3) protein from hepatitis C virus by chromatographic procedures. Biotechnol Lett 25:1729–1734PubMedGoogle Scholar
  169. 169.
    Schlegl R, Iberer G, Machold C et al (2003) Continuous matrix-assisted refolding of proteins. J Chromatogr A 1009:119–132PubMedGoogle Scholar
  170. 170.
    Zouhar J, Nanak E, Brzobohatý B (1999) Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific β-glucosidase. Protein Expr Purif 17:153–162PubMedGoogle Scholar
  171. 171.
    Rehm BHA, Qi Q, Beermann BB et al (2001) Matrix assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxylalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli. Biochem J 358:263–268PubMedGoogle Scholar
  172. 172.
    Cho TH, Ahn SJ, Lee EK (2001) Refolding of protein inclusion bodies directly from E. coli homogenate using expanded bed adsorption chromatography. Bioseparation 10:189–196PubMedGoogle Scholar
  173. 173.
    Li M, Zhang G, Su Z (2002) Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. J Chromatogr A 959:113–120PubMedGoogle Scholar
  174. 174.
    Negro A, Onisto M, Grassato L et al (1997) Recombinant human TIMP-3 from Escherichia coli: Synthesis, refolding physico-chemical and functional insights. Protein Eng 10:593–599PubMedGoogle Scholar
  175. 175.
    Stempfer G, Höll-Neugebauer B, Rudolph R (1996) Improved refolding of an immobilized fusion protein. Nat Biotechnol 14:329–334PubMedGoogle Scholar
  176. 176.
    Berdichevsky Y, Lamed R, Frenkel D et al (1999) Matrix-assisted refolding of single-chain Fv-cellulose binding domain fusion proteins. Protein Expr Purif 17:249–259PubMedGoogle Scholar
  177. 177.
    Suttnar J, Dyr JE, Hamšíková E et al (1994) Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger. J Chromatogr B 656:123–126Google Scholar
  178. 178.
    Geng X, Chang X (1992) High-performance hydrophobic interaction chromatography as a tool for protein refolding. J Chromatogr A 599:185–194Google Scholar
  179. 179.
    Bai Q, Kong Y, Geng X (2003) Studies on renaturation with simultaneous purification of recombinant human pro-insulin from E. coli with high performance hydrophobic interaction chromatography. J Liq Chromatogr Relat Technol 26:683–695Google Scholar
  180. 180.
    Gong B, Wang L, Wang C et al (2004) Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application. J Chromatogr A 1022:33–39PubMedGoogle Scholar
  181. 181.
    Jacquet A, Daminet V, Haumont M et al (1999) Expression of a recombinant Toxoplasma gondiiROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation. Protein Expr Purif 17:392–400PubMedGoogle Scholar
  182. 182.
    Martinez A, Knappskog PM, Olafsdottir S et al (1995) Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem J 306:589–597PubMedGoogle Scholar
  183. 183.
    Davis GD, Elisee C, Newham DM et al (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol Bioeng 65:382–388PubMedGoogle Scholar
  184. 184.
    Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674PubMedCrossRefGoogle Scholar
  185. 185.
    Sørensen HP, Sperling-Petersen HU, Mortensen KK (2003) A favorable solubility partner for the recombinant expression of streptavidin. Protein Expr Purif 32:252–259PubMedGoogle Scholar
  186. 186.
    Cabantous S, Waldo GS (2006) In vivo and in vitro protein solubility assays using split GFP. Nat Methods 3:845–854PubMedGoogle Scholar
  187. 187.
    Philipps B, Hennecke J, Glockshuber R (2003) FRET-based in vivo screening for protein folding and increased protein stability. J Mol Biol 327:239–249PubMedGoogle Scholar
  188. 188.
    Arechaga I, Miroux B, Runswick MJ et al (2003) Overexpression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript. FEBS Lett 547:97–100PubMedGoogle Scholar
  189. 189.
    Lee NP, Tsang S, Cheng RH et al (2006) Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag. Protein Pept Lett 13:431–435PubMedGoogle Scholar
  190. 190.
    Kim JS, Valencia CA, Liu R et al (2007) Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles. Bioconjug Chem 18:333–341PubMedGoogle Scholar
  191. 191.
    Hammarstrom M, Woestenenk EA, Hellgren N et al (2006) Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein. J Struct Funct Genomics 7:1–14PubMedGoogle Scholar
  192. 192.
    Feeney B, Soderblom EJ, Goshe MB et al (2006) Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker. Protein Expr Purif 47:311–318PubMedGoogle Scholar
  193. 193.
    Klein S, Geiger T, Linchevski I et al (2005) Expression and purification of active PKB kinase from Escherichia coli. Protein Expr Purif 41:162–169PubMedGoogle Scholar
  194. 194.
    de Marco A (2006) Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins. Nat Protoc 1:1538–1543PubMedGoogle Scholar
  195. 195.
    Cabrita LD, Dai W, Bottomley SP (2006) A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnol 6:12PubMedGoogle Scholar
  196. 196.
    Panagabko C, Morley S, Neely S et al (2002) Expression and refolding of recombinant human alpha-tocopherol transfer protein capable of specific alpha-tocopherol binding. Protein Expr Purif 24:395–403PubMedGoogle Scholar
  197. 197.
    Donath MJ, de Laaf RT, Biessels PT et al (1995) Characterization of des-(741–1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa. Biochem J 312:49–55PubMedGoogle Scholar
  198. 198.
    Prachayasittikul V, Isarankura Na Ayudhya C, Piacham T et al (2003) One-step purification of chimeric green fluorescent protein providing metal-binding avidity and protease recognition sequence. Asian Pac J Allergy Immunol 21:259–267PubMedGoogle Scholar
  199. 199.
    Kapust RB, Waugh DS (2000) Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr Purif 19:312–318PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sudhir Sahdev
    • 1
  • Sunil K. Khattar
    • 1
  • Kulvinder Singh Saini
    • 1
  1. 1.Department of Biotechnology & Bioinformatics, New Drug Discovery ResearchRanbaxy Research Laboratories-R&D-3GurgaonIndia

Personalised recommendations