Expression in Bacteria and Purification of Recombinant Proteins

A CaaX motif (Cys-Val-lle-Met) can be introduced C-terminally into the protein in question by standard techniques. The cDNA obtained can be cloned into an expression vector for E. coli, such as pTrc-99A (Pharmacia), and then transfected into E. coli DH5 a and induced with 5 mM isopropyl-(3-D-thioglactopyranoside (IPTG). The protein is isolated from the bacterial pellet. 

For purification of aFGF-CaaX, the bacterial pellet was suspended in 20 mM Tris-HCI, pH 7.5, 0.5 M NaCI, 1 mM DTT, 1 mM EDTA and sonicated. The clear supernatant, after centrifugation, was applied to a heparin cartridge (Bio-Rad), and the bound material was eluted with a linear NaCI gradient (0.5-2 M) in the above buffer. 

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