Phospholipid-detergent Micelles Research Articles

TheNeisseria meningitidisouter membrane protein P1 produced inBacillus subtilisand reconstituted into phospholipid vesicles elicits antibodies to native P1 epitopes

Class 1 outer membrane protein (P1) ofNeisseria meningitidisgroup B is considered a promising vaccine candidate because P1 subtype–specific antibodies have been shown to be protective in an animal model. We have previously described the production of P1 in the Gram–positiveBacillus subtilisas intracellular inclusion bodies, from which the protein (BacP1) is easily purified (Nurminenet al., Mol. Microbiol., 1992, 2499–2506). We show here that the purified BacP1 can be reconstituted into phospholipid vesicles with the formation of the native immunodominant surface epitopes. The detergent–solubilized, completely denatured BacP1 was fused with phospholipid–detergent micelles during detergent removal by dialysis or gel filtration to yield protein–lipid vesicles (liposomes). When mice were immunized with these liposomes, they produced high titers of antibodies reacting in a P1 subtype–specific manner with meningococcal cells indicating the presence of conformation–dependent P1–specific epitopes in the liposomes. The results suggest that a vaccine candidate for meningococcal disease could be developed from the BacP1–liposomes. They furthermore demonstrate the feasibility of refolding a denatured outer membrane protein, which has never been exposed to lipopolysaccharide, into a native–like conformation.

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.