Extra Leader Sequence Affects Immunoactivity of Cardiac Troponin I

Cardiac troponins are considered the preferred cardiac markers for diagnosis of acute myocardial infarction (1). The development and evaluation of cardiac troponin I immunoassays require purified and immunochemically stable antigen for the production of calibrators and controls. Cardiac troponin I (cTnI) purified from human heart has traditionally been used for such purposes. However, cTnI is unstable and prone to degradation during the process of purification from human heart tissue. Recombinant protein is favored over the protein purified from tissue because it is easier to purify, safer to handle, and has unlimited availability and good reproducibility. In physiological conditions, troponin C (TnC) and TnI along with troponin T (TnT) form an integral protein, designated as troponin complex. To study the immunoactivity of TnI in a clinically relevant form, it is imperative to put TnI in the context of the other two subunits, TnC and TnT. In this study, human cTnI, cTnC, and cTnT cDNAs were amplified from the Human Heart Quick-Clone cDNA (Clontech), using gene-specific primers designed from published cDNA sequences (2)(3)(4)(5) by PCR. Initially, the cDNA sequence for human cTnI was inserted into a pET vector without modification. Very little cTnI was expressed with this construct. To increase the expression of human cTnI in bacterial host cells, three different modifications were made to the cDNA sequences separately. The first modified clone, designated as rcTnI-0, was prepared with two NH2-terminal codon mutations without changing any amino acid residues, as described by Al-Hillawi et al. (6). The second and third modified clones, designated as rcTnI-6 and rcTnI-14, were constructed with added bacteria-favored NH2-terminal leader sequences (7); the leader sequences for rcTnI-6 and rcTnI-14 are MASMGS and MASMTGGQQMGRGS, respectively. The human cTnT cDNA sequence was modified as described previously (8) to increase its expression in Escherichia coli. TnC was expressed without modification to the cDNA sequence. All constructs were confirmed by DNA sequencing. Expression constructs for each protein were engineered, transformed, and expressed according to Shi et al. (9). The purification processes for these expressed proteins were essentially described by Shi et al. (9). The concentrations of all troponin subunits after purification were determined by Bradford assay (10), with bovine serum albumin as the calibrator. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on all samples according to Laemmli (11). As shown in Fig. 1 , human cardiac troponin subunits C, T, and I (I-0, I-6, and I-14) were successfully expressed in E. coli and purified to homogeneity. The apparent molecular weights of troponin I-0, I-6, and I-14 were ∼29 000, 30 000, and 31 000, respectively.