| Autor: |
Wu QL; Department of Anatomy and Cellular Biology, School of Veterinary Medicine, Tufts University, Boston, MA 02111, USA., Jha PK, Du Y, Leavis PC, Sarkar S |
| Jazyk: |
angličtina |
| Zdroj: |
Gene [Gene] 1995 Apr 03; Vol. 155 (2), pp. 225-30. |
| DOI: |
10.1016/0378-1119(94)00846-k |
| Abstrakt: |
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 beta TpnT-encoding cDNA (beta TpnTf) 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 beta TpnTf polypeptide from any species using an in vitro expression system. Purified human beta TpnTf, 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 alpha TpnT, a predominant adult isoform. These results provide a biochemical correlate to the age-related differences in Ca2+ sensitivity of tension development in vertebrate fast skeletal muscles. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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