[Fusion expression, purification and bioactivity detection of human defensinalpha (HDalpha)].
OBJECTIVE
To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research.


METHODS
The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection.


RESULTS
After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria.


CONCLUSIONS
The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.
Fu
Hai-yan
HY
Department of Biochemstry and Molecular Biology, Preclinical School, Fourth Military Medical University, Xi'an 710032, China.
Zhang
Jian
J
Lu
Fan
F
Pu
Qin
Q
Wang
Xiao-gong
XG
Lu
Zi-fan
ZF
Xu
Jun-qing
JQ
Zhao
Zhong-liang
ZL
D043169:Endostatins / Q000378:metabolism; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D023084:alpha-Defensins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D043169:Endostatins; D011993:Recombinant Fusion Proteins; D023084:alpha-Defensins; D004765:Enteropeptidase