Application of tandem affinity purification technique for separation of GroEL complexes in Escherichia coli

Abstract:

Enterohemorrhagic Escherichia coli O157:H7 is a kind of important pathogenic bacterium, and deeper studies of molecular pathogenesis will contribute better to related vaccine research and disease control. Tandem affinity purification (TAP) technique is a kind of new method which was recently developed and used to separate and purify native protein complexes and so on to study interactions between proteins. In this study, a prokaryotic TAP expression vector constructed by our laboratory was successfully used to express fusion protein GroEL-TAP in Enterohemorrhagic Escherichia coli O157:H7. An efficient method for preparation of native protein complexes was developed and all experimental parameters of tandem affinity purification were optimized. The highly pure protein complexes com-posed of both tagged GroEL-TAP and natural GroEL were obtained with our expression and purification system. These results showed that our TAP system worked well to specifically purify protein complex participated with target protein in Enterohemorrhagic Escherichia coli O157:H7. This study provided satisfactory experimental base for follow-on works on identifying virulence protein participated com-plexes.