Overproduction and rapid purification of human fast skeletal beta troponin T using Escherichia coli expression vectors: functional differences between the alpha and beta isoforms.
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.
Wu
Q L
QL
Department of Anatomy and Cellular Biology, School of Veterinary Medicine, Tufts University, Boston, MA 02111, USA.
Jha
P K
PK
Du
Y
Y
Leavis
P C
PC
Sarkar
S
S
D001483:Base Sequence; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014336:Troponin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D020107:Troponin T
D018076:DNA, Complementary; D014336:Troponin; D020107:Troponin T