PRODUCTION OF HAEMOPHILUS-INFLUENZAE TYPE-B PORIN IN ESCHERICHIA-COLIAND ITS FOLDING INTO THE TRIMERIC FORM

The P2 protein from pathogenic Haemophilus influenzae type b (Hib) fun ctions as a bacterial porin and is one of several immunogenic outer me mbrane proteins. The P2 gene was expressed in Escherichia coli and the recombinant P2 protein (re-P2) purified to facilitate functional and immunologic studies. P2 was obtained from Hib strain Eagan using PCR a nd the pET vectors (17b and 11a) were used to produce re-P2 at levels exceeding 30% of the total E. coli proteins. Since previous reports ha d indicated that P2 was toxic to E. coli, steps were taken to control the toxicity. The plasmid was stabilized by tightly controlling the sy nthesis of re-P2 prior to induction, Subsequent to induction, re-P2 wa s sequestered into inclusion bodies rather than to membrane compartmen ts. The refolding of the denatured re-P2 into the trimeric form involv ed high salt and calcium ions. re-P2 was then purified to homogeneity using gel-filtration and ion-exchange chromatography.