Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli and their purification and characterisation. Two point mutations allow high-level expression of troponin-I.
We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG-->GCC and (Gly4) GGG-->GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-I-troponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-S1 Mg-ATPase activity; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.
al-Hillawi
E
E
School of Biochemistry, University of Birmingham, England.
Minchin
S D
SD
Trayer
I P
IP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016296:Mutagenesis; D009206:Myocardium / Q000737:chemistry*; D015879:Myosin Subfragments / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014336:Troponin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I
D018076:DNA, Complementary; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I