[Expression, purification and identification of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27)].
OBJECTIVE
To construct prokaryotic expression vector of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27) and express the protein in E.coli.


METHODS
The rhLT-alphaDeltaN27 gene was amplified by RT-PCR using total RNA extracted from Jurkat cells,cloned into prokaryotic expression vector pET-23b, and transformed into E.coli BL21(DE3). The recombinant protein was expressed after IPTG induction and purified by DEAE Sepharose FF and Phenyl-Sepharose FF.


RESULTS
The recombinant protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein. After purification, the purity of rhLT-alphaDeltaN27 was 99%, and the biological activity was more than 8x10(7) U/mg. Other characteristics of rhLT-alphaDeltaN27, such as relative molecular mass(M(r)), pI and N-terminal amino acid sequence, all corresponded to theoretical prediction.


CONCLUSIONS
The expression vector of rhLT-alphaDeltaN27 gene was constructed, and the recombinant protein was expressed in E.coli successfully.A method of for purifying rhLT-alphaDeltaN27 was established.
Liu
Yong-quan
YQ
Beijing Tri-prime Genetic Engineering Co. Ltd., Beijing 102600, China. yongquanl@yahoo.com
Li
Dao-feng
DF
Niu
Xiao-xia
XX
Liu
Jin-yi
JY
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D008233:Lymphotoxin-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins