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Gene constructs for expression of recombinant proteins in microorganisms

The aim was to produce and verify constructs for expression of snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) in microorganisms to produce gram quantities of purified recombinant protein for feeding studies.

The Pichia pastoris strain X33 was transformed with pPICZB expression vector containing the structural gene for PHA-E, including the sequence encoding the signal peptide. After transformation, transformants were selected for zeocin resistance.

Similarly, the Pichia pastoris strain X33 was transformed with pPICZB expression vector containing the structural gene for GNA, including the sequence encoding the signal peptide. After transformation, transformants were selected for zeocin resistance.

The DNA construct (pOS4301) for expression of Cry1Ab was obtained from the Bacillus Genetic Stock Centre (BGSC) at the Ohio State University, USA (BGSC No. ECE54). The construct was assembled by Ge et al. (1989). Briefly, a wild-type full-length Bacillus thuringiensis Cry1Ab cDNA was cloned into vector pKK223-3 (Pharmacia), which contains the inducible tac promoter (Ptac) and transformed into Escherichia coli strain JM103.

Gram quantities of the recombinant proteins were produced by large-scale fermentation.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Contact

Angharad GATEHOUSE, (Director of Research, School of Biology)
Tél.: +44-191-2464811
Fax: +44-191-2225228
E-mail