[Genetic engineering in the bacterial synthesis of somatostatin].
Series of recombinant plasmids for expression of the synthetic gene somatostatin-14 (SST) as a fusion protein were obtained. The somatostatin gene was fused to chloramphenicol acetyltransferase (cat) or its deleted variant genes. Both parts of the resultant fusion protein were joined through a Met residue. The hybrid gene was expressed under the control of the cat gene promoter (Pcat), the tryptophan operon promoter (Ptrp) or the promoter of bacteriophage T5 (PT5). These fusions gave insoluble polypeptide products amounting from 5-10% of the total cellular protein under constitutive biosynthetic conditions (Pcat) to 5-30% upon induction (Ptrp, PT5). A correlation between the efficiency of expression and the length of cat, the power of the promoter used and the absence or presence of transcription terminators, was studied. The scheme for SST isolation from bacterial cells was developed. SST was liberated from the fused polypeptide by treatment with cyanogen bromide and purified to homogenity by a combination of chromatographic steps: gel filtration, ion-exchange and rpHPLC. The renaturated recombinant SST showed specific biological and immunological activities and had 98% purity. The yield was 1 mg of the purified cyclic SST/1 culture of E.coli.
Karpova
S K
SK
Sazina
E T
ET
Bader
L B
LB
Sergienko
O V
OV
Khodun
M L
ML
Krivtsov
V F
VF
Lunin
V G
VG
Tikhonenko
T I
TI
Pankov
Iu A
IuA
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics*; D015202:Protein Engineering; D013004:Somatostatin / Q000096:biosynthesis / Q000235:genetics*
D013004:Somatostatin; D015500:Chloramphenicol O-Acetyltransferase
Genno-inzhenernye podkhody k bakterialńomu syntezu somatostatina.