Cloning and hyperexpression of a gene encoding the heat-stable toxin of Escherichia coli.
A gene (st) coding for heat-stable toxin (STh) was identified from a plasmid of a locally isolated enterotoxigenic Escherichia coli strain. The gene was cloned and its nucleotide (nt) sequence was determined. Comparison of this nt sequence with that of another st gene reported earlier, showed a single nt substitution within the structural gene for ST. This change resulted in the replacement of proline at position 19 by alanine in the STh of the locally isolated strain. The st gene was hyperexpressed using the phage T7 or the tac promoter vector systems. A 20-fold increase in STh yield was obtained in minimal medium culture supernatants following induction of the T7 promoter. There was no significant accumulation of the precursor peptide within the periplasm of the induced cell, indicating efficient processing under conditions of enhanced transcription of the gene. The yield of STh was monitored using a competitive ELISA, which was found to be a simple and sensitive assay for determining STh concentrations. A rapid and efficient isolation procedure for STh has been developed.
Dwarakanath
P
P
Astra Research Centre India, Bangalore.
Visweswariah
S S
SS
Subrahmanyam
Y V
YV
Shanthi
G
G
Jagannatha
H M
HM
Balganesh
T S
TS
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli)