Expression of C. Elegans TnC Protein

In the spring of 1999 I worked on an independent student research project as part of the Cell Physiology Research class that I was taking at the time. This work was done under the able direction of Taylor Allen (Assistant Professor of Biology, Oberlin College) and was part of an ongoing project to purify two isoforms of the Troponin C (TnC) protein from C. Elegans. TnC contains the Ca²⁺ binding domain of the Troponin complex (Troponin C, I, and T) that regulate the interactions of Myosin and Actin necessary for muscle contraction. This is important because mutations in the Troponin complex are often linked to muscle diseases like Hypertrophic Cardial Myopathy.

My work started with the identification of the TnC isoforms contained in two pET-34b(+) plasmids with which two sets of E. Coli had already been transformed. This involved making DNA "mini-preps" from each strain and performing a digestion with EcoRV and SacI on the plasmids from the strains. The digested plasmid segments were then separated by agarose gel electrophoresis and stained with ethidium bromide. This tentatively identified which plasmid contained the gene for each TnC isoform (genes F54c1.7 and ZK763tnc respectively).

Subsequently, a test expression of the two TnC genes was done using BL21(DE3) and BL21(DE3)-pLysS E. Coli strains, which had been transformed with plasmids (previously assembled at this lab) containing the TnC isoforms. The transformed cells were incubated under both inducing and non-inducing conditions, with regards to production of the proteins encoded by the TnC genes in the pET34b(+) plasmids. Induction was caused by inclusion of IPTG in the growth medium. Each expressed TnC isoform also contained a cellulose binding domain for use in protein purification. After incubation, whole cell lysates from the protein expression cells were separated on a discontinuous polyacrylamide gel and stained with Coomassie blue. This test expression showed significant production of both TnC isoforms by the BL21(DE3) cells, while little or no expression was observed in the BL21(DE3)-pLysS cells which have "tighter" regulation of induction. Future work on this project would include scaling up protein production and the purification of TnC isoforms for analysis of their Ca²⁺ binding dynamics.

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Copyright 2001-2006 Ben Coburn	Last Updated: 1:31 May 3, 2006