%0 Journal Article
%T Cloning of mMR-1 Gene and Expression in Pichia pastoris Systems<br>mMR-1基因的克隆和在毕赤酵母中分泌表达
%A LI Tian Bo
%A HU Yang
%A WANG Yi Guang
%A XIA Huan Zhang Institute of Medicinal Biotechnoly
%A CAMS & PUMC
%A Beijing
%A China Shenyang Pharmaceutical University
%A Shenyang
%A China
%A <br>李天伯
%A 胡洋
%A 王以光
%A 夏焕章
%J 生物工程学报
%D 2005
%I 
%X hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.
%K MR-1
%K cloning and expression
%K Pichia pastoris<br>MR-1
%K 克隆与表达
%K 毕赤酵母
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=C6A3A0EBBA0498A1&yid=2DD7160C83D0ACED&vid=659D3B06EBF534A7&iid=CA4FD0336C81A37A&sid=C5154311167311FE&eid=771469D9D58C34FF&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=11