Expression of Recombinant Alkaline Phosphatase Conjugates in Escherichia coli

Abstract

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA. The presence of an intact PhoA signal peptide yields to a systematic secretion of the fusion proteins into the periplasm where the PhoA dimerises to its active form, and disulfides can be formed if necessary. The resultant PhoA-tagged proteins are particularly convenient novel tools that can be used in a wide range of applications, including expression, epitope mapping, histochemistry, immunoblotting, mutant analysis, and competition or sandwich ELISAs (see Note 1). Expression of an scFv antibody fragment derived from an IgG2a/κ immunoglobulin specific for curaremimetic toxins from snake (named M-α2-3), will be used to illustrate the methods utilized for its cloning, expression in E.coli, extraction, and functional characterization.