Applied Microbiology and Biotechnology

, Volume 72, Issue 2, pp 211–222 | Cite as

Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems

  • Kay TerpeEmail author


During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial systems remain most attractive due to low cost, high productivity, and rapid use. However, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. This review gives an overview of the most commonly used systems: As hosts, Bacillus brevis, Bacillusmegaterium, Bacillussubtilis, Caulobacter crescentus, other strains, and, most importantly, Escherichia coli BL21 and E. coli K12 and their derivatives are presented. On the promoter side, the main features of the l-arabinose inducible araBAD promoter (PBAD), the lac promoter, the l-rhamnose inducible rhaPBAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter pL, and the anhydrotetracycline-inducible tetA promoter/operator are summarized.


Recombinant Protein Codon Usage Codon Usage Bias Bacterial Host Promoter System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The author thanks Prof. A. Steinbüchel for supporting this review.


  1. Amann E, Brosius J, Ptashne M (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167–178PubMedGoogle Scholar
  2. Anna DF, Rosa M, Emilia P, Simonetta B, Mose R (2003) High-level expression of Aliciclobacillus acidocaldarius thioredoxin in Pichiapastoris and Bacillus subtilis. Protein Expr Purif 30:179–184Google Scholar
  3. Arechaga I, Miroux B, Karrasch S, Huijbregts R, De Kruiff B, Runswick MJ, Walker JE (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F1F0 ATP synthase. FEBS Lett 482:215–219PubMedGoogle Scholar
  4. Armarego WL, Cotton RG, Dahl HH, Dixon NE (1989) High-level expression of human dihydropteridine reductase (EC, without N-terminal amino acid protection, in Escherichia coli. Biochem J 261:265–268PubMedPubMedCentralGoogle Scholar
  5. Awram P, Smit J (1998) The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus. J Bacteriol 180:3062–3069PubMedPubMedCentralGoogle Scholar
  6. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421PubMedGoogle Scholar
  7. Barnard GC, Henderson GE, Srinivasan S, Gerngross TU (2004) High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification. Protein Expr Purif 38:264–271PubMedGoogle Scholar
  8. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA 96:13703–13708PubMedGoogle Scholar
  9. Bingle WH, Nomellini JF, Smit J (2000) Secretion of the Caulobacter crescentus S-layer protein: Further localization of the C-terminal secretion signal and its use for secretion of recombinant proteins. J Bacteriol 182:3298–3301PubMedPubMedCentralGoogle Scholar
  10. Braaz R, Wong SL, Jendrossek D (2002) Production of PHA depolymerase A (PhaZ5 from Paucimonas lemoignei in Bacillus subtilis. FEMS Microbiol Lett 209:237–241PubMedGoogle Scholar
  11. Brawner ME (1994) Advances in heterologous gene expression by Streptomyces. Curr Opin Biotechnol 5:475–481PubMedGoogle Scholar
  12. Brosius J, Erfle M, Storella J (1985) Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity. J Biol Chem 260:3539–3541PubMedGoogle Scholar
  13. Burger S, Tatge H, Hofmann F, Genth H, Just I, Gerhard R (2003) Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system. Biochem Biophys Res Commun 307:584–588PubMedGoogle Scholar
  14. Cho HY, Yukawa H, Inui M, Doi RH, Wong S-L (2004) Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl Environ Microbiol 70:5704–5707PubMedPubMedCentralGoogle Scholar
  15. Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635PubMedPubMedCentralGoogle Scholar
  16. Daguer JP, Chamber R, Petit-Glatron MF (2005) Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis. Lett Appl Microbiol 41:221–226PubMedPubMedCentralGoogle Scholar
  17. De Boer HA, Comstock LJ, Vasser M (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80:21–25PubMedPubMedCentralGoogle Scholar
  18. Degenkolb J, Takahashi M, Ellestad GA, Hillen W (1991) Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor. Antimicrob Agents Chemother 35:1591–1595PubMedPubMedCentralGoogle Scholar
  19. DeLisa MP, Li J, Rao R, Weigand WA, Bentley WE (1999) Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor. Biotechnol Bioeng 65:54–64PubMedPubMedCentralGoogle Scholar
  20. Desplanq D, Bernard C, Sibler AP, Kieffer B, Miguet L, Potier N, Van Dorsselaer A, Weiss E (2005) Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120. Biotechniques 39:405–411Google Scholar
  21. Dong H, Nilsson L, Kurland CG (1996) Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rate. J Mol Biol 260:649–663Google Scholar
  22. 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
  23. Egan SM, Schleif RF (1993) A regulatory cascade in the induction of rhaBAD. J Mol Biol 234:87–98PubMedGoogle Scholar
  24. Elvin CM, Thompson PR, Argall ME, Hendry P, Stamford NP, Lilley PE, Dixon NE (1990) Modified bacteriophage lambda promoter for overproduction of proteins in Escherichia coli. Gene 87:123–126PubMedPubMedCentralGoogle Scholar
  25. England DF, Penfold RJ, Delaney SF, Rogers PL (1997) Isolation of Bacillus megaterium mutants that produce high levels of heterologous protein, and their use to construct a highly mosquitocidal strain. Curr Microbiol 35:71–76PubMedGoogle Scholar
  26. FitzGerald KA, Lidstrom ME (2003) Overexpression of a heterologous protein, haloalkane dehalogenase, in a poly-beta-hydroxybutyrate-deficient strain of the facultative methylotroph Methylobacterium extorquens AM1. Biotechnol Bioeng 81:263–268PubMedGoogle Scholar
  27. Geissendörfer M, Hillen W (1990) regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements. Appl Microbiol Biotechnol 33:657–663PubMedGoogle Scholar
  28. Georgiou G, Segatori L (2005) Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr Opin Biotechnol 16:538–545PubMedGoogle Scholar
  29. Goldman E, Rosenberg AH, Zubay G, Studier FW (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
  30. Gronenborn (1976) Overproduction of phage lambda repressor under control of the lac promotor of Escherichia coli. Mol Gen Genet 148:243–250PubMedGoogle Scholar
  31. Grossman TH, Kawasaki ES, Punreddy SR, Osburne MS (1998) Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene 209:95–103PubMedGoogle Scholar
  32. Gutierrez J, Bourque D, Criado R, Choi YJ, Cintas LM, Hernandez PE, Miguez CB (2005) Heterologous extracellular production of enterocin P from Enterococcus faecium P13 in the methylotrophic bacterium Methylobacterium extorquens. FEMS Microbiol Lett 248:125–131PubMedGoogle Scholar
  33. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedPubMedCentralGoogle Scholar
  34. Haldimann A, Daniels L, Wanner B (1998) Use of new methods for construction of tightly regulated arabinose and rhamnose promotor fusions in studies of the Escherichia coli phosphate regulon. J Bacteriol 180:1277–1286PubMedPubMedCentralGoogle Scholar
  35. Hansson M, Samuelson P, Nguyen TN, Stahl S (2002) General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae. FEMS Microbiol Lett 210:263–270PubMedGoogle Scholar
  36. Henner DJ (1990) Inducible expression of regulatory genes in Bacillus subtilis. Methods Enzymol 185:223–228PubMedGoogle Scholar
  37. Hockney RC (1994) Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol 12:456–463PubMedPubMedCentralGoogle Scholar
  38. Horinouchi S, Weisblum B (1982) Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol 150:804–814PubMedPubMedCentralGoogle Scholar
  39. Huang HC, Sherman MY, Kandror O, Goldberg AL (2001) The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli. J Biol Chem 276:3920–3928PubMedGoogle Scholar
  40. Ichikawa Y, Yamagata H, Tochikubo K, Udaka S (1993) Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis. FEMS Microbiol Lett 111:219–224PubMedGoogle Scholar
  41. Inoue Y, Ohta T, Tada H, Iwasa S, Udaka S, Yamagata H (1997) Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis. Appl Microbiol Biotechnol 48:487–492PubMedGoogle Scholar
  42. Joly JC, Swartz JR (1994) Protein folding activities of Eschrichia coli protein disulfide isomerase. Biochemistry 12:4231–4236Google Scholar
  43. Kajino T, Saito Y, Hirai M, Asami O, Yamada Y, Udaka S (1997) Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system. J Ind Microbiol Biotech 19:227–231Google Scholar
  44. Kajino T, Ohto C, Muramatsu M, Obata S, Udaka S, Yamada Y, Takahashi H (1999) A protein disulfid isolerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis. Appl Environ Microbiol 66:638–642Google Scholar
  45. Kajino T, Takahashi H, Hirai M, Yamada Y (2000) Efficient production of artificially designed gelatins with a Bacillus brevis system. Appl Environ Microbiol 66:304–309PubMedPubMedCentralGoogle Scholar
  46. Kane JF (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6:494–500PubMedPubMedCentralGoogle Scholar
  47. Kashima Y, Udaka S (2004) High-level production of hyperthermophilic cellulase in the Bacillus brevis expression and secretion system. Biosci Biotechnol Biochem 68:235–237PubMedGoogle Scholar
  48. Kawamura F, Doi RH (1984) Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol 160:442–444PubMedPubMedCentralGoogle Scholar
  49. Kim L, Mogk A, Schumann W (1996) A xylose-inducible Bacillus subtilis interation vector and its application. Gene 181:71–76PubMedGoogle Scholar
  50. Konishi H, Sato T, Yamagata H, Udaka S (1990) Efficient production of human alpha-amylase by a Bacillus brevis mutant. Appl Microbiol Biotechnol 34:297–302PubMedGoogle Scholar
  51. Korpela MT, Kurittu JS, Karvinen JT, Karp MT (1998) A recombinant Escherichia coli sensor strain for the detection of tetracyclines. Anal Chem 70:4457–4462PubMedGoogle Scholar
  52. Kurland C, Gallant J (1996) Errors of heterologous protein expression. Curr Opin Biotechnol 7:489–493PubMedGoogle Scholar
  53. Lam KH, Chow KC, Wong WK (1998) Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins. J Biotechnol 63:167–177PubMedGoogle Scholar
  54. Landry TD, Chew L, Davis JW, Frawley N, Foley HH, Stelman SJ, Thomas J, Wolt J, Hanselmann DS (2003) Safety evaluation of an α-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I. Regul Toxicol Pharmacol 37:149–168PubMedGoogle Scholar
  55. Le Grice SF (1990) Regulated promoter for high-level expression of heterologous genes in Bacillus subtilis. Methods Enzymol 185:201–214PubMedGoogle Scholar
  56. Lesuisse E, Schanck K, Colson C (1993) Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extreme basic pH-tolerant enzyme. Eur J Biochem 216:155–160Google Scholar
  57. Li W, Zhou X, Lu P (2004) Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol 155:605–610PubMedGoogle Scholar
  58. Love CA, Lilley PE, Dixon NE (1996) Stable high-copy-number of bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene 176:49–53PubMedGoogle Scholar
  59. Lowman HB, Bina M (1990) Temperature-mediated regulation and downstream inducible selection for controlling gene expression from the bacteriophage lambda pL promoter. Gene 96:133–136PubMedGoogle Scholar
  60. Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC7l1–l2 regulatory elements. Nucleic Acids Res 25:1203–1210PubMedPubMedCentralGoogle Scholar
  61. Makrides SC (1996) Strategies for achieving high level expression of genes in Escherichia coli. Microbiol Rev 60:512–538PubMedPubMedCentralGoogle Scholar
  62. Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218PubMedPubMedCentralGoogle Scholar
  63. Margot P, Karamata D (1996) The wrpA gene Bacillus subtilis 168 expressed during exponential growth, encodes a cell-wall-associated protease. Microbiology 142:3437–3444PubMedGoogle Scholar
  64. Mayer M (1995) A new set of useful cloning and expression vectors derived from pBlueScript. Gene 163:41–46PubMedGoogle Scholar
  65. McKenzie T, Hoshino T, Tanaka T, Sueoka N (1986) The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15:93–103PubMedGoogle Scholar
  66. Meinhardt F, Stahl U, Eveling W (1989) Highly efficient expression of homologous and heterologous genes in Bacillus megaterium. Appl Microbiol Biotechnol 30:343–350Google Scholar
  67. Menart V, Jevsevar S, Vilar M, Trobis A, Pavko A (2003) Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda. Biotechnol Bioeng 83:181–190PubMedGoogle Scholar
  68. Mergulhao FJ, Monteiro GA (2004) Secretion capacity limitations of the Sec pathway in Escherichia coli. J Microb Biotechnol 14:128–133Google Scholar
  69. Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202PubMedGoogle Scholar
  70. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high level. J Mol Biol 260:289–298PubMedPubMedCentralGoogle Scholar
  71. Moffatt BA, Studier FW (1987) T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell 49:221–227PubMedGoogle Scholar
  72. Murashima K, Chen C-L, Kosugi A, Tamaru Y, Doi RH, Wong S-L (2002) Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. J Bacteriol 184:76–81PubMedPubMedCentralGoogle Scholar
  73. Nakamura K, Furusato T, Shiroza T, Yamane K (1985) Stable hyper-production of Escherichia coli β-lactamase by Bacillus subtilis grown on a 0.5 M succinate-medium using a B. subtilis α-amylase secretion vector. Biochem Biophys Res Commun 128:601–606PubMedGoogle Scholar
  74. Olmos-Soto J, Contreras-Flores R (2003) Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis. Appl Microbiol Biotechnol 62:369–373PubMedGoogle Scholar
  75. Palva I (1982) Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19:81–87PubMedGoogle Scholar
  76. Palva I, Lehtovaara P, Kaariainen L, Sibakov M, Cantell K, Schein CH, Kashiwagi K, Weissmann C (1983) Secretion of interferon by Bacillus subtilis. Gene 22:229–235PubMedGoogle Scholar
  77. Pan SH, Malcolm BA (2000) Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). Biotechniques 29:1234–1238PubMedGoogle Scholar
  78. Petsch D, Anspach FB (2000) Endotoxin removal from protein solutions. J Biotechnol 76:97–119PubMedGoogle Scholar
  79. Phillips TA, Van Bogelen RA, Neidhardt FC (1984) lon gene product of Escherichia coli is a heat-shock protein. J Bacteriol 159:283–287PubMedPubMedCentralGoogle Scholar
  80. Polisky B, Bishop RJ, Gelfand DH (1976) A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria. Proc Natl Acad Sci USA 73:3900–3904PubMedGoogle Scholar
  81. Puyet A, Sandoval H, Lopez P, Aguilar A, Martin JF, Espinosa M (1987) A simple medium for rapid regeneration of Bacillus subtilis protoplasts transformed with plasmid DNA. FEMS Microbiol Lett 40:1–5Google Scholar
  82. Quick M, Wright EM (2002) Employing Escherichia coli to functionally express, purify, and characterize a human transporter. Proc Natl Acad Sci USA 99:8597–8601PubMedGoogle Scholar
  83. Ritz D, Lim J, Reynolds CM, Poole LB, Beckwith J (2001) Conversion of a peroxiredoxin into a disulfide reductase by a triplet repeat expansion. Science 294:158–160PubMedGoogle Scholar
  84. Rosenberg HF (1998) Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques 24:188–191PubMedGoogle Scholar
  85. Rygus T, Hillen W (1991) Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of xylose-utilization operon. Appl Microbiol Biotechnol 35:594–599PubMedGoogle Scholar
  86. Rygus T, Scheler A, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol 155:535–542PubMedGoogle Scholar
  87. Sagiya Y, Yamagata H, Udaka S (1994) Direct high-level secretion into the culture medium of tuna growth hormone in a biologically active form by Bacillus brevis. Appl Microbiol Biotechnol 42:358–363PubMedGoogle Scholar
  88. Schmidt FR (2004) Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol 65:363–372PubMedPubMedCentralGoogle Scholar
  89. Schneider JC, Jennings AF, Mun DM, McGovern PM, Chew LC (2005) Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation. Biotechnol Prog 21:343–348PubMedGoogle Scholar
  90. Shiga Y, Maki M, Ohta T, Tokishita S, Okamoto A, Tsukagoshi N, Udaka S, Konishi A, Kodama Y, Ejima D, Matsui H, Yamagata H (2000) Efficient production of N-terminally truncated biologically active human interleukin-6 by Bacillus brevis. Biosci Biotechnol Biochem 64:665–669PubMedGoogle Scholar
  91. Shokri A, Sande’n AM, Larsson G (2003) Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 60:654–664PubMedGoogle Scholar
  92. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310PubMedGoogle Scholar
  93. Skerra A (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131–135Google Scholar
  94. Stahl ML, Ferrari E (1984) Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro derived deletion mutation. J Bacteriol 158:411–418PubMedPubMedCentralGoogle Scholar
  95. Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219:37–44PubMedGoogle Scholar
  96. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130PubMedPubMedCentralGoogle Scholar
  97. Takimura Y, Kato M, Ohta T, Yamagata H, Udaka S (1997) Secretion of human interleukin-2 in biologically active form by Bacillus brevis directly into culture medium. Biosci Biotechnol Biochem 61:1858–1861PubMedGoogle Scholar
  98. Tjalsma H, Bolhuis A, Jongbloed JD, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547PubMedPubMedCentralGoogle Scholar
  99. Udaka S, Yamagata H (1993) High-Level secretion of heterologous proteins by Bacillus brevis. Methods Enzymol 217:23–33PubMedGoogle Scholar
  100. Ulmanen K, Lundstrom P, Lehtovaara P, Sarvas M, Ruohonen M, Palva I (1985) Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. J Bacteriol 162:176–182PubMedPubMedCentralGoogle Scholar
  101. Umelo-Njaka E, Nomellini JF, Yim H, Smit J (2001a) Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus. Plasmid 46:37–46PubMedGoogle Scholar
  102. Umelo-Njaka E, Nomellini JF, Bingle WH, Glasier LG, Irvin RT, Smit J (2001b) Expression and testing of Pseudomonasaeruginosa vaccine candidate proteins prepared with the Caulobactercrescentus S-layer protein expression system. Vaccine 19:1406–14015PubMedPubMedCentralGoogle Scholar
  103. Vary PS (1994) Prime time for Bacillus megaterium. Microbiology 140:1001–1013PubMedGoogle Scholar
  104. Vorobjeva IP, Khmel LA, Alfödi L (1980) Transformation of Bacillus megaterium protoplasts by plasmid DNA. FEMS Microbiol Lett 7:261–263Google Scholar
  105. Wang RF, Kushner SR (1991) Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195–199PubMedGoogle Scholar
  106. Wang B, Yang X, Wu R (1993) High-level production of the mouse epidermal growth factor in a Bacillus brevis expression system. Protein Expr Purif 4:223–231PubMedGoogle Scholar
  107. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as a cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694:299–310PubMedGoogle Scholar
  108. Wilms B, Hauck A, Reuss M, Syldatk C, Mattes R, Siemann M, Altenbuchner J (2001) High-cell-density fermentation for production of l-N-Carbamoylase using an expression system based on the Escherichia colirhaBAD promoter. Biotechnol Bioeng 73:95–103PubMedPubMedCentralGoogle Scholar
  109. Wong S-L, Kawamura F, Doi RH (1986) Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM β-lactamase during growth. J Bacteriol 168:1005–1009PubMedPubMedCentralGoogle Scholar
  110. Wu S-C, Wong S-L (2002) Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin. Appl Environ Microbiol 68:1102–1108PubMedPubMedCentralGoogle Scholar
  111. Wu S-C, Wong S-L (2005) Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem 280:23225–23231PubMedGoogle Scholar
  112. Wu X-C, Lee W, Tran L, Wong S-L (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173:4952–4958PubMedPubMedCentralGoogle Scholar
  113. Wu S-C, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68:3261–3269PubMedPubMedCentralGoogle Scholar
  114. Yamagata H, Nakahama K, Suzuki Y, Kakinuma A, Tsukagoshi N, Udaka S (1989) Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA 86:3589–3593PubMedGoogle Scholar
  115. Yang S, Huang H, Zhang R, Huang X, Li S, Yuan Z (2001) Expression and purification of extracellular penicillin G acylase in Bacillus subtilis. Protein Expr Purif 21:60–64Google Scholar
  116. Ye R, Kim JH, Kim BG, Szarka S, Sihota E, Wong S-L (1999) High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnol Bioeng 62:87–96PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.IBA GmbHGöttingenGermany

Personalised recommendations