[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor].
Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.
Korobko
V G
VG
Boldyreva
E F
EF
Filippov
S A
SA
Berkova
N P
NP
Dobrynin
V N
VN
Shmelev
V A
VA
Popov
S G
SG
Evsegneev
S I
SI
Nosova
L Iu
LIu
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005347:Fibroblasts; D015870:Gene Expression; D005813:Genes, Synthetic; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013947:Thymosin; D000077596:Thymalfasin
Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholeĭ cheloveka.