Overexpression of Human Cardiac Troponin‐I and Troponin‐C in Escherichia coli and Their Purification and Characterisation

Abstract

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-Sl MgATPase 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.Troponin and tropomyosin, located on the actin thin filament of vertebrate skeletal and cardiac muscles, are responsible for the calcium-dependent regulation of the actomyosin Mg-ATPase activity [l]. Troponin is a complex of three subunits ; troponin-C (Tn-C), the calcium-binding component, troponin-I (Tn-I), the inhibitory subunit, and troponin-T (Tn-T), which locates the troponin complex on the tropomyosin (TM) complex. Tn-C undergoes a conformational change upon the binding of calcium; this is transmitted through the troponin complex, weakening the Tn-I-actin interaction and causing TM to move away from the myosin-binding sites on actin [2]. The result is the formation of actomyosin crossbridges at these sites, the hydrolysis of Mg-ATP and force generation [3, 41. Cardiac and skeletal isoforms of Tn-I are highly similar in their sequences. The cardiac isoform differs from its skeletal counterpart in possessing a 30-33-amino-acid (speciesdependent) N-terminal extension [ 5 ] . This contains the cardiac-specific adjacent phosphorylation sites at Ser23-Ser24 in human and bovine sequences [6, 71. There is good evidence that this phosphorylation is involved in the modulaCorrespondence to I. P. Trayer,

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

“…2). Although a previous study saw improved expression of human cardiac TnI in E. coli by modifying codons Ala 2 and Gly 4 [25], our modification of Gly 4 and Arg 10 codons did not reproduce the improvement in cardiac TnI expression as that seen in the tRNA-enhanced E. coli cells (Fig. 3E and Table 1).…”

“…Such improvement was also seen by others [25]. To quantitatively investigate the incompatibility effects of cardiac TnI N-terminal region on bacterial expression, we compared the expression of an N-terminal truncated mouse cardiac TnI lacking amino acids 1-28.…”

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin‐I mutants and troponin‐C

“…To synthesize Ser23Asp/Ser-24Asp, the following oligonucleotide was used: 5'-GCG GTA GTT ATC GTC GCG GCG TCT GAT-3';this introduced aspartic acid residues at positions 23 and 24 to replace the serines. Both genes were cloned into the pET1 lc vector as previously described [19]. The DNA sequence of the two mutants was checked by the dideoxy chain termination method [28].…”

“…These were subsequently used to transform competent BL21(DE3) cells. Protein expression as described by A1-Hillawi et al (1994) [19].The two mutant proteins were purified using ion-exchange chromatography [19] except that a 0-0.5 M NaCI gradient was used to elute the protein of the CM-Sepharose fast flow column.…”

“…The phosphorylation of cardiac TnI has been shown to reduce the affinity of TnC for Ca 2+ [18], thus, promoting more rapid relaxation [9]. Different TnI isoforms from various tissues have been overexpressed in bacterial systems [19,20] and a number of mutants have been produced in order to investigate the structur~func-tion relationship in TnI [7,10,[21][22][23]. Here, we report, for the first time, the reconstitution into skinned cardiac fibres of recombinant human cardiac TnC and Tnl which will provide a convenient test system for investigating engineered forms of these proteins.…”