Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA.
The ospA gene of Borrelia burgdorferi encodes an outer membrane protein which is a major antigen of the Lyme disease agent. Two sequence-specific sets of oligonucleotide primers were used to specify the amplification of the ospA coding sequence by the polymerase chain reaction. One set allowed the entire ospA sequence to be amplified, while the other primed amplification of a truncated form of ospA lacking the first 17 codons specified by the wild-type ospA structural gene, residues believed to constitute a signal sequence which normally would direct localization of the ospA protein to the Borrelia cell's outer membrane. Each set of primers also contained sequences near their 5' ends which facilitated cloning of the amplified DNA directly into a high level expression system based on bacteriophage T7 genetic elements. We showed that the full-length OspA protein is synthesized poorly in Escherichia coli and it is associated with the insoluble membrane fraction. In contrast, the truncated form can be expressed to very high levels and it is soluble. The truncated protein was purified to homogeneity and partially characterized. Its N-terminal sequence and molecular weight derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis agree with those deduced from the DNA sequence. It is a monomer with a native molecular weight of 28,000 and it is very resistant to digestion by trypsin even though it is rather rich in lysine residues (16 mol%). Recombinant OspA protein synthesized in E. coli is recognized by antibodies in sera of Lyme patients, which suggests that the protein may be useful in immunoassays and as a possible immunogen to protect against Lyme borreliosis.
Dunn
J J
JJ
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Lade
B N
BN
Barbour
A G
AG
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines; D001483:Base Sequence; D025065:Borrelia burgdorferi; D015748:Borrelia burgdorferi Group / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D008074:Lipoproteins; D008193:Lyme Disease / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D008074:Lipoproteins; C072045:OspA protein; D011994:Recombinant Proteins