Expression and hydroxylamine cleavage of thymosin alpha 1 concatemer.
Human thymosin alpha 1 (Talpha1) is an important peptide in the development and senescence of immunological competence in human, and many studies have reported the expression of this peptide. In this study, we designed and synthesized the Talpha1 gene according to the E. coli codon usage preference and constructed a 6xTalpha1 concatemer. The latter was inserted into an E. coli expression vector pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG, the concatemer protein was successfully expressed in E. coli then cleaved by hydroxylamine to release the Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry confirmed that the recombinant protein was cleaved as intended. The bioactivity of the Talpha1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines. This study provides a description of the preparation of a bioactive Talpha1, which may prove useful in future biomedical research.
Zhou
Liang
L
Room 345, College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
Lai
Zong-Teng
ZT
Lu
Min-Kan
MK
Gong
Xing-Guo
XG
Xie
Yi
Y
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D005562:Formazans / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D019811:Hydroxylamine / Q000494:pharmacology*; D002479:Inclusion Bodies / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005562:Formazans; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013778:Tetrazolium Salts; C079782:MTT formazan; D019811:Hydroxylamine; D007544:Isopropyl Thiogalactoside; D013947:Thymosin; D000077596:Thymalfasin