Prakash K. Jha

Overproduction and rapid purification of human fast skeletal β troponin T using Escherichia coli expression vectors: functional differences between the α and β isoforms

Abstract Troponin T (TpnT), an essential component of the Ca 2+ -regulatory troponin complex, is involved in protein-protein interactions with other thin-filament proteins during muscle contraction in vertebrate striated muscle (VSM). The isoforms of TpnT are encoded by members of a multigene family which, by alternate splicing, produces a complex pattern of isoproteins in VSM. The functional domains of TpnT are only tentatively identified and structure-function analysis on this protein is limited due to the heterogeneity of the multiple isoforms. We reasoned that the overproduction and purification of a single TpnT species in Escherichia coli would provide an insight into these studies, besides being useful in crystallizing the protein. We cloned the human fast skeletal αTpnT-encoding cDNA (βTpnT f ) in three expression vectors. Overexpression was achieved in an E. coli BL21(DE3) lysogen using a T7 RNA polymerase promoter-based vector, pET17b. The unfused recombinant protein was purified by a simple and rapid procedure in a biologically active and immunoreactive form. This is the first successful synthesis of a complete βTpnT f polypeptide from any species using an in vitro expression system. Purified human βTpnT f , a predominant fetal form, was less Ca 2+ -sensitive and exhibited considerably reduced affinity for troponin C and tropomyosin, as compared to the rabbit fast skeletal αTpnT, a predominant adult isoform. These results provide a biochemical correlate to the age-related differences in Ca 2+ sensitivity of tension development in vertebrate fast skeletal muscles