Isolation, cloning, and overexpression of a chitinase gene fragment from the hyperthermophilic archaeon Thermococcus chitonophagus: semi-denaturing purification of the recombinant peptide and investigation of its relation with other chitinases.
A 189-bp sequence was isolated from the hyperthermophilic archaeon Thermococcus chitonophagus and was found to present strong homology with a large number of chitinase genes from a variety of organisms and particularly with the chitinaseA gene from Pyrococcus kodakaraensis (Pk-chiA). This fragment was subcloned to an expression vector and overexpressed in Escherichia coli. The E. coli BLR21(DE3)pLysS transformant, harbouring the gene on the pET-31b plasmid vector, was found to overproduce the target protein at high levels. The 63 aminoacid-long peptide was efficiently purified to homogeneity, with a one-step, semi-denaturing affinity chromatography, on a metal chelation resin and was used for the production of a specific, polyclonal antibody from rabbits. The produced antibody was demonstrated to display strong and specific affinity for the chitinase A from Serratia marcescens (Sm-chiA), as well as the membrane-bound chitinase70 from Thermococcus chitonophagus (Tc-Chi70). The strong sequence homology, in combination with the demonstrated specific immunochemical affinity, indicates that the isolated peptide is part of a chitinolytic enzyme of T. chitonophagus. In particular, it could belong to the membrane-bound chi70, or to a distinct chitinase, coded by a different gene, or even by the same gene, following post-transcriptional or post-translational modifications.
Andronopoulou
Evi
E
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15701 Athens, Greece.
Vorgias
Constantinos E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D019641:DNA, Archaeal; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019641:DNA, Archaeal; D011994:Recombinant Proteins; D002688:Chitinases