Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta 8 outer membrane protein Omp21 from Comamonas acidovorans

The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta -strands which form a beta -barrel . This family includes virulence proteins, OmpA and OmpX from Escherichia c oli, and other related molecules. After we established an expression system , recombinant Omp21 was purified by Ni2+ chelation affinity chromatography and refolded in situ while bound to resin, The native state of refolded pro tein was proven by FTIR spectroscopy and monitored with denaturing PAGE (he at modification). Both native and recombinant Omp21 were reconstituted in l ipid membranes and crystallized two-dimensionally by controlled dialysis. R ecombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D struc ture of negatively stained, recombinant Omp21 was determined at a resolutio n of 1.8 nm by means of electron crystallography, Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity bet ween the mass distribution of exoplasmic loops of Omp21 and OmpA. (C) 2000 Academic Press.