Cloning and expression of the Propionibacterium shermanii methylmalonyl-CoA epimerase gene in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
Genomic DNA was isolated from Propionibacterium shermanii (52W). A 454 bp DNA fragment coding for the methylmalonyl-CoA epimerase (EC 126.96.36.199, subsequently referred to as epimerase) was amplified from genomic DNA by the polymerase chain reaction using primers designed from the known DNA sequence of the gene. The P. shermanii epimerase gene was ligated into the 2.47 kbp expression vector pT7-7. The ligation reaction mixture was transformed into electroporation competent E.coli XL1-Blue cells. Plasmid DNA prepared from several transformants was analysed, by agarose gel electrophoresis of restriction enzyme digestions, and transformed into E.coli SRP84/pGP1-2 cells to identify potential epimerase expression constructs (pTEEX) by heat shock induction. The insert DNA of one of the putative pTEEX epimerase constructs was fully sequenced and shown to be identical to the known DNA sequence of the epimerase gene described by Davis (1987). Using the sequenced expression construct pTEEX, recombinant epimerase was expressed to 20-35% of the total cell protein in the protease deficient E.coli strain SRP84 using the dual plasmid expression system of Tabor and Richardson (1985). The recombinant epimerase was ~95-100% soluble in E.coli. The recombinant epimerase and the 'wild-type' epimerase produced by P. shermanii were purified using the procedures developed for the 'wild-type' epimerase. The addition of a heat-treatment step (70°C for 15 min) early in the purification of the recombinant enzyme successfully exploited the unusually high thermostability of the epimerase protein. The epimerase protein was found to have an anomalously low electrophoretic mobility in a modified Laemmli discontinuous Tris-glycine alkaline buffer system for SDS-PAGE gels compared to the Weber and Osborn continuous phosphate buffer system. Using the latter system, a subunit molecular weight of 16.6 kDa was obtained. This is consistent with the molecular weight of 16.72 kDa (methionine on) calculated from the inferred amino acid sequence. The N-terminal sequence of the purified 'wild-type' and recombinant epimerases were identical although only half of N-terminal methionine residues were removed from the recombinant protein. The subunit molecular weight, specific activity, activation by divalent metal ions and behaviour in crystallization trials of the 'wild-type' and recombinant epimerases were very similar. Recombinant epimerase crystals were grown in a buffer containing 0.2 M ammonium acetate and 0.1 M citrate, pH 5.6, containing 30% PEG 4000 as precipitant. These crystals were relatively poorly ordered and diffracted to only 4.5 Ǻ resolution, but crystals of the recombinant epimerase that diffract to 2.6Ǻ can be grown under appropriate conditions.