Cloning of mMR-1 gene and expression in Pichia pastoris systems.
- Author:
Tian-Bo LI
1
;
Yang HU
;
Yi-Guang WANG
;
Huan-Zhang XIA
Author Information
1. Insitute of Medicinal Biotechnology, CAMS & PUMC, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Cloning, Molecular;
Humans;
Mice;
Mice, Inbred C57BL;
Molecular Sequence Data;
Muscle Proteins;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Plasmids;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Reverse Transcriptase Polymerase Chain Reaction
- From:
Chinese Journal of Biotechnology
2005;21(1):25-29
- CountryChina
- Language:Chinese
-
Abstract:
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.
