[Cloning of mMR-1 gene and expression in Pichia pastoris systems].
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.
Li
Tian-Bo
TB
Insitute of Medicinal Biotechnology, CAMS & PUMC, Beijing 100050, China.
Hu
Yang
Y
Wang
Yi-Guang
YG
Xia
Huan-Zhang
HZ
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009124:Muscle Proteins; C495444:PNKD protein, human; D011994:Recombinant Proteins