PREPARATION OF Sry/pIVEX BACTERIAL EXPRESSION CONSTRUCTS AND RECOMBINANT PROTEIN EXPRESSION.

Sry is a gene on the Y chromosome that is responsible for testes formation during development. The Sry protein acts as a transcription factor. Sry is also expressed in adult male tissues, which suggests the possibility of additional functions. There are six unique Sry loci present in Rattus norvegicus, and the functions of these are yet to be identified. The objective of this project was to insert each of the six rat Sry genes into the pIVEX2.4 expression plasmid and collect the recombinant protein for use in future Sry research. An N-terminal histidine tag encoded by the pIVEX2.4 vector allowed for one-step purification of the Sry protein by using Ni sepharose 6 fast flow beads (GE Healthcare). All six rat Sry genes were successfully cloned into pIVEX2.4. All constructs were transformed into Lucigen BL21(DE) E. coli and induced for 2 hours with .75mM IPTG at a cell density of 0.8 at OD600. Collection and subsequent analysis of recombinant protein by SDS-PAGE confirmed expression from Sry2/pIVEX2.4A and Sry3/pIVEX2.4A clones. These two Sry proteins are currently being used in gel shift assays to study its role as a transcription factor. [ABSTRACT FROM AUTHOR]