RAO Jing-jing, JING Yi-xian, ZOU Ming-yue, HU Xiao-lei, LIAO Fei, YANG Xiao-lan
Objective:To clone the uricase gene of the Meyerozyma guilliermondii(C.G.M.C.C 2.1008) for recombinant expression in Escherichia coli and further characterization. Methods:The strain was identified by sequences of 28S rRNA D1/D2 and ITS. Partial peptide sequences were recognized through trypsin digestion and MS/MS analyses. The induction expression of the uricase was verified by RNA-seq. The target gene was amplified by PCR through precise primers designed according to the coding sequence of a homologus putative uricase of Meyerozyma guilliermondii ATCC 6260. The cloned coding sequence was inserted into the expression vectors pDE1 and pDE2 to construct the plasmids pDE1-MGU and pDE2-MGU bearing 6His tags. After the deletion of 6His tag and the linking peptide, the vector for the non-tagged MGU was constructed and denoted R-MGU. The plasmids were transformed into E. coli BL21 (DE3) for induced expression. The recombinant protein was characterizedby SDS-PAGE, MALDI-TOF-MS and the related enzymatic properties. Result:The sequences of rRNA supported that it was a strain of Meyerozyma guilliermondii. MS/MS analyses supported its high homology to a putative uricase A5DFP1 deduced from the genomic sequence of Meyerozyma guilliermondii ATCC 6260 and its induced expression by uric acid was further verified by RNA-seq. After PCR cloning with a pair of precise primers, the coding sequence of the targeted MGU showed the only different base at the 435th site but gave the same amino acid residue. After recombinant expression, R-MGU had the peptide weight of about 35kDa by SDS-PAGE, but of 17.43 by MALDI-TOF-MS that was consistent with that of the wildtype, supporting the unidentified chemical modification of MGU. The maximum specific activity of the recombinant form of the uricase was about 6.0U/mg. Of R-MGU, the Km, Ki of representative inhibitors and molecular weight had no significantly differences from the wildtype. However, the thermostability of R-MGU was slightly worse than the wildtype, due primarily to the low purity of the sample. Conclusion:From Meyerozyma guilliermondii(C.G.M.C.C 2.1008), MGU gene was cloned and expressed successfully as the active enzyme in Escherichia coli.
