[Expression and purification of recombinant Trail protein in E.coli and the optimal conditions for Trail purification].
OBJECTIVE
To construct the expression vector pET-28α-Trail(114-281); and find the optimal conditions for target gene expression,  host bacteria lysis,  and protein purification, and to  detect the apoptosis function of the recombinant  protein.


METHODS
The functional domain of Trail(114-281); was amplified by PCR and  cloned into the expression vector pET-28α(+). After confirmed by DNA sequencing,  the Trail(114-281);   was expressed in E.coli BL21 under the condition of different A(600);,  IPTG concentration   and temperature. Host bacteria were lysed using three different ways,  including ultrasonication, osmotic shock and IP lysis, and  the target protein was purified using Ni-NTA affinity chromatography or cutting-gel purification. The advantages and shortcomings of these methods were compared to find  the most efficient ways for  expression and purification of the recombinant protein.  The immunocompetence of Trail protein from cutting-gel purification  was analyzed by Western blotting,   A549 cell apoptosis induced by purified protein from Ni-NTA chromatography  was detected by flow cytometry.


RESULTS
The 516 bp Trail(114-281); gene was cloned,  and expressed in E.coli BL21.  When  A(600);=0.6, recombinant host bacteria were  induced by 1.0 mmol/L IPTG  at 37 DegreesCelsius  for 4 h,  which was the optimal condition  for the expression of inclusion body,  and the soluble protein was expressed stably on the condition of 25 DegreesCelsius, A(600);=1.0,  IPTG1.0 mmol/L. Ultrasonication could get maximal  protein compared to the other methods. The two purification ways both could purify taget protein successfully. Western blot analysis showed that the protein purified by cutting-gel has  a good immunologic activity. Protein from Ni-NTA affinity chromatography  caused cell apoptosis.


CONCLUSIONS
The expression vector pET-28α-Trail(114-281); can be   constructed and expressed in E.coli  BL21 successfully.  Temperature is a more important effect factor of Trail(114-281); expression in host bacteria compared with other factors. Cutting-gel protein has  immunogenicity,  and Ni-NTA protein could keep its function. These results provide a  basis  for the further    functional research and application of Trail.
Department of Clinical Laboratory, Guangzhou Medical College, Guangzhou, China.
Yuan-yuan
YY
Cai
Xue-yan
XY
Li
Yu
Y
Wu
Jing-chun
JC
DU
Xia
X
Xu
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand