[Soluble expression, purification and bioactivity of recombinant human Era protein].
OBJECTIVE
To prepare a soluble human Era(hEra) protein and to measure its bioactivity.


METHODS
Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG).


RESULTS
The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP.


CONCLUSIONS
Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.
Wu
Yuan-ming
YM
Center for DNA Typing, Fourth Military Medical University, Xi'an, China. wuym@fmmu.edu.cn
Zhang
Xiao-nan
XN
Xin
Xiao-hong
XH
Zhang
Jun-jie
JJ
Chen
Nan-chun
NC
Chen
Su-min
SM
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
C474774:ERas protein, human; D011993:Recombinant Fusion Proteins; D015689:Oncogene Protein p21(ras)