Expression of recombinant alkaline phosphatase conjugates in Escherichia coli.
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. Expression of an scFv antibody fragment derived from an IgG2a/kappa immunoglobulin specific for curaremimetic toxins from snake (named M-alpha2-3), will be used to illustrate the methods utilized for its cloning, expression in E.coli, extraction, and functional characterization.
Département d'Ingénierie et d'Etudes des Protéines, CEA Saclay, Gif-sur-Yvette Cedex, France.
Jean-Claude
JC
Boulain
Frédéric
F
Ducancel
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase