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 allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.
Nguyen
T N
TN
Centre d'Immunologie Pierre Fabre, F-74 164, Saint-Julien en Genevois, France.
Samuelson
P
P
Sterky
F
F
Merle-Poitte
C
C
Robert
A
A
Baussant
T
T
Haeuw
J F
JF
Uhlén
M
M
Binz
H
H
Stâhl
S
S
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D001483:Base Sequence; D019921:Biotinylation / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013205:Staphylococcal Protein A / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A