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Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli

  • Usama BeshayEmail author
  • Gerhard Miksch
  • Karl Friehs
  • Erwin Flaschel
Original Paper

Abstract

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a β-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn2+-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml−1 PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher β-glucanase concentration (65.6 kU ml−1), i.e. 1.5-fold compared to the internal adsorbent system.

Keywords

Integrated process Production and purification E. coli β-glucanase 

List of symbols

X

mass concentration (g/l)

ρL,ext

extracellular glucanase activity (U/ml)

ρL,ads

adsorbed glucanase activity on resin (U/ml)

ρL,∑

total glucanase activity (U/ml)

t

time of cultivation (h)

Notes

Acknowledgments

Dr. U. Beshay acknowledges the financial support from the Alexander von Humboldt Foundation for a visiting fellowship to Bielefeld University, Germany.

References

  1. 1.
    Arvidsson P, Plieva FM, Lozinsky VI, Galsev IY, Mattiasson B (2003) Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent. J Chromatogr A 986:275–290CrossRefGoogle Scholar
  2. 2.
    Wang HY, Kominek LA, Jost JL (1981) On-line extraction fermentation processes. In: Moo-Young M, Robinson CW, Vezina C (eds) Advances in biotechnology I. Scientific and engineering principles, vol 1. Pergamon Press, Oxford, pp 601–607Google Scholar
  3. 3.
    Wang HY, Robinson FM, Lee SS (1981) Biotechnol Bioeng Symp 11:555–565Google Scholar
  4. 4.
    Wang HY (1983) Integrating biochemical separation and purification steps in fermentation processes. Ann NY Acad Sci 413:313–321CrossRefGoogle Scholar
  5. 5.
    Margaritis A, Bajpai P (1982) Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus. Biotech Bioeng 24:1483–1493CrossRefGoogle Scholar
  6. 6.
    Knoop B, Beiderbeck R (1983) Adsorbenskultur – ein Weg zur Steigerung der Sekundarstoffproduktion in pflanzlichen Suspensionskulturen. Z Naturforsch 38:484–486Google Scholar
  7. 7.
    Becker H, Reichling JH, Bisson W, Herold S (1984) Two phase culture—a new method to yield lipophilic secondary products from plant suspension cultures. Proceedings of the 3rd European Congress on biotechnology, vol 1, pp 209–213Google Scholar
  8. 8.
    Robins RJ, Rhodes MJ (1986) The stimulation of anthraquinone production by Cinchona ledgeriana cultures with polymeric adsorbents. Appl Microbiol Biotechnol 24:35–41CrossRefGoogle Scholar
  9. 9.
    Miksch G, Neitzel R, Fiedler E, Friehs K, Flaschel E (1997) Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors. Appl Microbiol Biotechnol 47:120–126CrossRefGoogle Scholar
  10. 10.
    Miksch G, Fiedler E, Dobrowolski P, Flaschel E (1997) Controlled secretion into the culture medium of a hybrid β-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon. Appl Microbiol Biotechnol 47:530–536CrossRefGoogle Scholar
  11. 11.
    Miksch R, Neitzel K, Friehs E, Flaschel (1999) High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium. Appl Microbiol Biotechnol 51:627–632CrossRefGoogle Scholar
  12. 12.
    Hochuli E, Bannwarth W, Doebeli H, Gentz R, Stueber D (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnology 6:1321–1325CrossRefGoogle Scholar
  13. 13.
    Arnold FH (1991) Metal-affinity separations: a new dimension in protein processing. Biotechnology 9:151–156CrossRefGoogle Scholar
  14. 14.
    Flaschel E, Friehs K (1993) Improvement of downstream processing of recombinant proteins by means of genetic engineering methods. Biotech Adv 11:31–78CrossRefGoogle Scholar
  15. 15.
    Calado CRC, Ferreira BS, Fonseca MMR, Cabral JMS, Fonseca LP (2004) Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain. J Biotechnol 109:147–158CrossRefGoogle Scholar
  16. 16.
    Ho LF, Li SY, Lin SC, Hsu WH (2004) Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents. Proc Biochem 39:1573–1581CrossRefGoogle Scholar
  17. 17.
    Kumar A, Bansal V, Nandakumar KS, Galaev IY, Roychoudhury PK, Holmdahl R, Mattiasson Bo (2006) Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices. Biotechnol Bioeng 93:636–646CrossRefGoogle Scholar
  18. 18.
    Charoenrat T, Ketudat-Cairns M, Jahic M, Enfors SO, Veide A (2006) Recovery of recombinant β-glucosidase by expanded bed adsorption from Pichia pastoris high-cell density culture broth. J Biotechnol 122:86–98CrossRefGoogle Scholar
  19. 19.
    Beshay U, Miksch G, Friehs K, Flaschel E (2007) Improved β-glucanase production by a recombinant Escherichia coli strain using zinc-ion supplemented medium. Eng Life Sci 7:253–258CrossRefGoogle Scholar
  20. 20.
    Miksch G, Kleist S, Friehs K, Flaschel E (2002) Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol 59:685–694CrossRefGoogle Scholar
  21. 21.
    Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Env Microbiol 64:82–87Google Scholar
  22. 22.
    Borriss R, Olsen O, Thomsen KKK, von Wettstein D (1989) Hybrid bacillus endo-(1–3, 1–4)- β-glucanases: construction of recombinant genes and molecular properties of the gene product. Carlsberg Res Commun 54:41–54CrossRefGoogle Scholar
  23. 23.
    Minh TQ (2006) The patented pentadentate chelator (PDC) is the best chelator in immobilized metal ion affinity chromatography (IMAC) for 6xhis-tagged protein purification and general purification of proteins. Affiland. Affinity methodology in biotechnology, pp 1–7Google Scholar
  24. 24.
    Beshay U, Friehs K, Azzam A, Flaschel E (2003) Cultivation of Dictyostelium discoideum in immobilized form by colonization of porous supports. Proc Biochem 38:1521–1529CrossRefGoogle Scholar
  25. 25.
    Beshay U, Miksch G, Flaschel E (2007) Improvement of a β-glucanase activity test by taking into account the batch reactor balance of the test system. Bioprocess Biosyst Eng 30:251–259CrossRefGoogle Scholar
  26. 26.
    Beshay U, Miksch G, Friehs K, Flaschel E (2003) Production of a bacterial β-glucanase by expression in Escherichia coli and simultaneous adsorption on a metal chelate affinity resin. Arab J Biotechnol 6:183–190Google Scholar
  27. 27.
    Bae J, Moon H, Oh KK, Kim CH, Lee DS, Kim SW, Hong SI (2001) A novel bioreactor with an internal adsorbent for integrated fermentation and recovery of prodigiosin-like pigment produced from Serratia Sp. KH-95. Biotechnol Lett 23:1315–1319CrossRefGoogle Scholar
  28. 28.
    Gupta M, Jain S, Roy I (2002) Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads. Biotechnol Prog 18:78–81CrossRefGoogle Scholar
  29. 29.
    Roe SD (1987) Whole broth extraction of enzymes from fermentation broths using commercially available adsorbents. In: Verrall MS, Hudson MJ (eds) Separations for biotechnology. Ellis Horwood, Chichester, pp 210–215Google Scholar
  30. 30.
    Mohan SB, Chohan SR, Eade J, Lyddian A (1993) Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery. Biotechnol Bioeng 42:974–986CrossRefGoogle Scholar
  31. 31.
    Born C, Thoemmes J, Biselli M, Wandrey C, Kula MR (1996) An approach to integrated antibody production: coupling of fluidized bed cultivation and fluidized bed adsorption. Bioprocess Eng 15:21–29CrossRefGoogle Scholar
  32. 32.
    Beer HD, Maschke HE, Schuegerl K (1992) Continuous production of restriction endonucleases: continuous two-stage cultivation with Escherichia coli JM103, continuous cell disintegration and purification by affinity chromatography. Appl Microbiol Biotechnol 38:220–225CrossRefGoogle Scholar
  33. 33.
    Gehael I, Kula MR (1984) Integration of ion exchange and ultrafiltration steps studied during purification of formate dehydrogenase using DEAE dextran. Biotechnol Lett 6:481–486CrossRefGoogle Scholar
  34. 34.
    Pungor E, Afeyan N, Gordon NF, Cooney CL (1987) Continuous affinity-recycle extraction: a novel protein separation technique. Biotechnology 5:604–608CrossRefGoogle Scholar
  35. 35.
    Hamilton GE, Luechau F, Burton SC, Lyddiatt A (2000) Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures. J Biotechnol 79:103–115CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Usama Beshay
    • 1
    Email author
  • Gerhard Miksch
    • 2
  • Karl Friehs
    • 2
  • Erwin Flaschel
    • 2
  1. 1.Bioprocess Development DepartmentMubarak City for Scientific Research and Technology Applications, Genetic Engineering and Biotechnology Research Institute (GEBRI)AlexandriaEgypt
  2. 2.Technische FakultaetUniversitaet BielefeldBielefeldGermany

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