title: Is it possible to optimize the protein production yield by the generation of homomultimeric fusion enzymes?
abstract: BACKGROUND
  The supply of industrially relevant biocatalysts demands an easy and efficient protein production in high yield. In a conventional approach, a recombinant protein is produced in a heterologous host enabling the manipulation of multiple parameters including expression plasmids, growth conditions and regulation of protein biosynthesis. In this study, the generation of homomultimeric fusion genes is tested as an additional parameter to increase the production yield of a heat-stable cellulase.
  
  
  RESULTS
  The LE (LguI/Eco81I)-cloning strategy was used to generate a set of plasmids containing a single copy or two to four repetitions of the endoglucanase-encoding gene cel5A from the thermophilic anaerobe Fervidobacterium gondwanense. Serial up-scaling of shaking flask volumes from 50 to 500 mL were used to determine the production yield of active cellulolytic enzyme Cel5A in recombinant form in Escherichia coli. Monitoring the cellular wet weight and total protein proved that the bacterial growth rate is not depending on the production of fusion enzymes, however activity assays in combination with Western blotting analyses indicated instability effects of large homomultimeric fusion enzymes.
  
  
  CONCLUSIONS
  The production yield of fusion cellulases is constant with increasing molecular weights, but improved activities were not observed for recombinant Cel5A homomultimers. This strategy may serve as a starting point for further studies to generate more stable fusion proteins with improved catalytic activities and higher protein yield in the future.
authors.affiliation: Institute for Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, 21073 Hamburg, Germany.
authors.forename: Iryna
authors.initials: I
authors.lastname: Barshakh
authors.affiliation: Institute for Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, 21073 Hamburg, Germany.
authors.forename: Skander
authors.initials: S
authors.lastname: Elleuche
keywords: Endoglucanase; Gene fusion; LE-cloning; Protein yield; Stability; Thermozymes