Expression of Pyrococcus abyssi recombinant alkaline phosphatase:: influences of Escherichia coli rare codons and secretion by the methylotrophic yeast Pichia pastoris

Alkaline phosphatases (APs) are non-specific phosphomonoesterases which are of interests for basic research as well as for practical applications. So far, Pyrococcus abyssi AP is the only AP which was isolated from a hyperthermophilic archaeon. In the present work, expression of this enzyme using mesophilic hosts was investigated. When R abyssi AP was produced by Escherichia coli using the regulation of the T7 promoter, only 0.6 mg of recombinant enzyme per liter of culture were obtained. Using the same system with the co-expression of tRNAs related to the rare codons AGA, AGG and ATA, the expression level was 14-fold higher. The methylotrophic yeast Pichia pastoris was also used to produce P. abyssi AP. A recombinant pPICZalphaA was constructed in order to have the AP secreted in the culture medium. A Pc. pastoris cell line producing P abyssi AP was studied in 1.21 working volume fermentor. Secretion was achieved with low expression yield but the recombinant enzyme appeared more active than when produced in E. coli. (C) 2003 Elsevier Science Inc. All rights reserved.