摘要
买述丁,一说西夏人,一说回回人,其在元朝政治舞台上活跃60余载,为政期间,革新吏治、清除积弊、裁减冗员、轻徭减役、赈济灾荒、救助百姓、整顿海运、重建漕府、抗击海寇等,为元代的社会发展作出了贡献,留下了功绩。本文通过对买述丁相关文献的比对研究,主要对其生平事迹作一简要考述。
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 ASDFP1 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/rag. Of R-MGU, the Kin, 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 guiUiermondii （ C. G. M. C. C 2. 1008）, MGU gene was cloned and expressed successfully as the active enzyme in Escherichia coll.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第11期74-82,共9页
China Biotechnology
基金
国家社会科学基金项目“西夏遗民文献整理与研究”（项目编号：13BTQ034）阶段性研究成果
关键词
买述丁
西夏人
回回人
事迹
Meyerozyma guilliermondii uricase Gene cloning Recombinant expression Characterization
