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Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.

Författare:
  • Anna Collén
  • Klaus Selber
  • Teppo Hyytiä
  • Josefine Persson
  • Tiina Nakari-Setlä
  • Michael Bailey
  • Richard Fagerström
  • Maria-Regina Kula
  • Merja Penttilä
  • Henrik Stålbrand
  • Folke Tjerneld
Publiceringsår: 2002
Språk: Engelska
Sidor: 385-394
Publikation/Tidskrift/Serie: Biotechnology and Bioengineering
Volym: 78
Nummer: 4
Dokumenttyp: Artikel i tidskrift
Förlag: John Wiley & Sons

Sammanfattning

Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.

Nyckelord

  • Biological Sciences

Övriga

Published
  • ISSN: 1097-0290

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