Expression of truncated and full-length forms of the Lyme disease Borrelia outer surface protein A in Escherichia coli.
The lipidated major outer surface protein, OspA, of the Lyme disease spirochaete may be important in the pathogenesis during Lyme borreliosis. To produce sufficient amounts of purified OspA variants to perform pathogenesis studies in vivo and in vitro, different recombinant OspA expression systems in Escherichia coli were constructed. Recombinant OspA variants were produced as a full-length molecule, as a truncated variant lacking the N-terminal lipidated cysteine, or as a fusion protein with the synthetic dimer of Staphylococcus aureus protein A IgG binding domain (ZZ). In order to produce the full-length protein, four different promoters were evaluated. These were combined with either the OspA original signal sequence or the E. coli Brauns lipoprotein signal sequence, lpp. The most efficient production of the full-length lipidated OspA was mediated by the constitutive beta-lactamase promoter in combination with lipoprotein signal sequences. For production of truncated nonlipidated OspA the S. aureus protein A signal sequence was ligated to the OspA open reading frame. Alternatively, truncated OspA was produced intracellularly using expression vectors that lack signal sequences. Production of nonlipidated protein with a heterologous signal peptide resulted in a soluble protein located mainly in the periplasm and in the culture medium. The full-length lipidated OspA, on the other hand, was associated mainly with the membrane fraction. The production level of the lipidated recombinant OspA was much lower than the level obtained with the truncated ZZ-OspA fusion protein.
Hansson
L
L
Symbicom AB, Umeå, Sweden.
Noppa
L
L
Nilsson
A K
AK
Strömqvist
M
M
Bergström
S
S
D000954:Antigens, Surface / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001428:Bacterial Vaccines; D001483:Base Sequence; D001898:Borrelia / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; D009838:Oligodeoxyribonucleotides; C072045:OspA protein; D011993:Recombinant Fusion Proteins