Cloning expression in E.coli and biological activity of human thymosin beta(4).
The cDNA thymosin beta(4) was synthesized by combining of chemical and enzymatic methods. First, two complement fragments of thymosin beta(4) cDNA were synthesized by DNA synthesizer, and then denatured, annealed and extended by DNA polymerase. This fragment of thymosin beta(4) was then inserted into the EcoRV and HindIII restriction endonuclease site of an expression plasmid pLDH4 (a kind of E.coli plasmid) by blunt and cohesive ligations. Finally, the recombinant plasmid which expressed thymosin beta(4) was screened by digestion and DNA sequencing. This recombinant plasmid highly expressed the thymosin beta(4), which accounted for 30% of total bacteria proteins. By salting out and chromatography, a 95% purity of recombinant thymosin beta(4) was obtained. Biological assay indicated that the recombinant thymosin beta(4) could induce lymphocyte proliferation and differentiation.
Che
Yan-Ke
YK
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Yang
Hui
H
Lu
Fan
F
Pu
Qin
Q
Li
Ren-De
RD
Zhao
Zhong-Liang
ZL
D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013947:Thymosin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000099201:Thymosin beta(4)
D011994:Recombinant Proteins; D013947:Thymosin; D000099201:Thymosin beta(4)