DESIGN OF A SOLUBILIZATION PATHWAY FOR RECOMBINANT POLYPEPTIDES IN-VIVO THROUGH PROCESSING OF A BI-PROTEIN WITH A VIRAL PROTEASE

An artificial maturation pathway for increasing the solubility in vivo of recombinant proteins overproduced in Escherichia coil is reported, which is based on the proteolytic processing of viral polyproteins. T he gene product of interest is expressed as a fusion to heterologous m oiety (i.e. the maltose binding protein, MalE) in order to increase th e overall solubility of the hybrid, The hinge region between the two f usion partners contains a cleavage site for the NIa protein, a very sp ecific protease from the plum pox potyvirus, as well as an affinity ta g, After production, the soluble hybrid is cleaved in vivo by the prot ease, that is encoded by a plasmid harboured by a specialized E.coli h ost, The released protein remains soluble and can be purified from cel l extracts by means of an affinity tag (a poly-His group) that becomes present after the cleavage, The solubilization and purification of Xy IR, a xylene-responsive transcriptional factor of Pseudomonas, with th is method are reported.