CHROMOSOMAL SEQUENCING USING A PCR-BASED BIOTIN-CAPTURE METHOD ALLOWED ISOLATION OF THE COMPLETE GENE FOR THE OUTER-MEMBRANE PROTEIN-A OF KLEBSIELLA-PNEUMONIAE

By employing a novel biotin-and PCR-assisted capture method, which all ows determination of unknown sequences on chromosomal DNA. the gene fo r the outer membrane protein A (OmpA) of Klebsiella pneumoniae has bee n isolated and sequenced to completion. The method involves linear amp lification of DNA from a biotinylated primer annealing to a region wit h known sequence. After capture of the amplified single-stranded DNA o n to paramagnetic beads, unspecifically annealing primers, i.e. arbitr ary primers, were used to generate fragments with only partly determin ed nt sequences. The homology of the sequenced gene to ompAs of relate d bacteria is discussed. The ompA gene was assembled for intracellular expression in Escherichia coli, and two different fusion proteins wer e produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the p otential use of the OmpA fusion proteins are discussed. (C) 1998 Elsev ier Science B.V.