[Expression and purification of truncated fragment of extracellular segment of sodium pump alpha3 subunit in Escherichia coli by in-fusion technology].
OBJECTIVE
To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology.


METHODS
According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump a3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-alpha3 (Truncated fragment of extracellular segment of sodium pump alpha3 subunit, Trf-alpha3). After DH10bac was transferred with it, the pGEX-Trf-alpha3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX-Trf-alpha3 was expressed in E. coli BL21 cells, inducted by IPTG. GST-Trf-alpha3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE.


RESULTS
The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-alpha3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33.22 X 10(3). The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80.8%. After affinity purification, the purity of GST-Trf-alpha3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-alpha3 fusion protein and ouabain, but the activity was very low.


CONCLUSIONS
Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been constructed. The purified method had also established. High purified GST-Trf-alpha3 fusion protein was obtained. These have found the foundation of further study on its biological function and potential pharmacology function.
Zhang
Ming-juan
MJ
Department of Cardiology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710004, China.
Yang
Jun
J
Zhu
Can-zhan
CZ
Duan
Zong-ming
ZM
Niu
Xiao-lin
XL
Wang
Rong
R
Song
Ya-fan
YF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C526097:Atp1a3 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase