Expression of repetitive human calcitonin genes in Escherichia coli.
In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E. coli under the control of a constitutive synthetic phage promoter. The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc. The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene). This effect held true as well for the proteins coded by the corresponding repetitive hCT genes. The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats. The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E. coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics). One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene. The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E. coli strains in order to find the optimal host for production of rhCT. The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.
Gigova
L
L
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
Wishart
P
P
Uscheva
A
A
Ivanova
M
M
Bardarov
S
S
Jay
E
E
Ivanov
I
I
D002116:Calcitonin / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D012333:RNA, Messenger; D011994:Recombinant Proteins; D002116:Calcitonin; D004247:DNA