Production of Haemophilus influenzae type-b porin in Escherichia coli and its folding into the trimeric form.
The P2 protein from pathogenic Haemophilus influenzae type b (Hib) functions as a bacterial porin and is one of several immunogenic outer membrane 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 and 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 had indicated that P2 was toxic to E. coli, steps were taken to control the toxicity. The plasmid was stabilized by tightly controlling the synthesis of re-P2 prior to induction. Subsequent to induction, re-P2 was sequestered into inclusion bodies rather than to membrane compartments. The refolding of the denatured re-P2 into the trimeric form involved high salt and calcium ions. re-P2 was then purified to homogeneity using gel-filtration and ion-exchange chromatography.
Pullen
J K
JK
North American Vaccine, Inc., Beltsville, MD 20705.
Liang
S M
SM
Blake
M S
MS
Mates
S
S
Tai
J Y
JY
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics*; D008969:Molecular Sequence Data; D018272:Porins / Q000096:biosynthesis* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D011994:Recombinant Proteins; C074703:outer membrane protein P2, Haemophilus influenzae type b