Recombinant outer-surface protein A (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi: high production levels in Saccharomyces cerevisiae yeast cultures.
The recombinant outer-surface protein A with an N-terminally truncated form (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi was expressed in Saccharomyces cerevisiae at high production levels. Since the recombinant vaccine candidate expressed in Escherichia coli exhibits low production yields and the purification of lipoproteins appears to be difficult, we have investigated the secretion of a soluble recombinant OspA in the yeast S. cerevisiae. In this way, a Leu+ derivative of S. cerevisiae cI3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding des-Cys1-OspA and driven by the MF alpha 1 promoter. The fed-batch culture results revealed that an efficient secretion of des-Cys1-OspA is obtained with a high production level of about 2.1 g l-1 at a cell density of 101 g l-1 cell dry weight. The accumulation of recombinant protein in the supernatant exceeds 6% of the total yeast proteins when estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Moreover, des-Cys1-OspA showed lower solubilities at high cell densities and, as a consequence, a fraction of the recombinant protein precipitated. An internal cleavage of the MF alpha 1 pro::des-Cys1-OspA precursor was also detected. However, in this case the cleavage occurred at a frequency such that the large amounts of the secreted des-Cys1-OspA could be employed for the evaluation of an immunogenic effect on animal immunization. These studies will extend the knowledge of the usefulness of OspA as a vaccine for Lyme borreliosis.
Mendoza-Vega
O
O
Keppi
E
E
Bouchon
B
B
Nguyen
M
M
Achstetter
T
T
D000954:Antigens, Surface / Q000096:biosynthesis*; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis*; D001428:Bacterial Vaccines; D025065:Borrelia burgdorferi; D015748:Borrelia burgdorferi Group / Q000737:chemistry*; D008074:Lipoproteins; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; C072045:OspA protein; D011994:Recombinant Proteins