Abstract
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 MFα1 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 1−1 at a cell density of 101 g 1−1 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 MFα1 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.
Similar content being viewed by others
References
Achstetter T, Nguyen-Juilleret M, Findeli A, Merkamm M, Lemoine Y (1992) A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin. Gene 110:25–31
Barbour AG, Heiland RA, Howe TR (1985) Heterogeneity of major proteins of Lyme disease borrelia: a molecular analysis of North American and European isolates. J Infect Dis 152:478–484
Bergström S, Bundoc VG, Barbour AG (1989) Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochete Borrelia burgdorferi. Mol Microbiol 3:479–486
Bouchon B, Van Dorsselaer A, Roitsch C (1993) Characterization of multiple acylation sites on a recombinant protein by electrospray mass spectrometry. Biol Mass Spectrometry 22:358–360
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Dunn JJ, Lade BN, Barbour AG (1990) Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA. Prot Exp Purif 1:159–168
Edelman R (1991) Perspective on the development of vaccines against Lyme disease. Vaccine 9:531–532
Erdile LF, Brandt MA, Warakomski DJ, Westrack GJ, Sadziene A, Barbour AG, Mays JP (1993) Role of attached lipid in immunogenieity of Borrelia burgdorferi OspA. Infect Immunol 61:81–90
Fiechter A, Fuhrmann GF, Käppeli O (1981) Regulation of glucose metabolism in growing yeast cells. Adv Microb Phys 22:123–183
Fikrig E, Barthold SW, Kantor FS, Flavell RA (1990) Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 250:553–556
Fikrig E, Telford SR III, Barthold SW, Kantor FS, Spielman A, Flavell RA (1992) Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice. Proc Natl Acad Sci USA 89:5418–5421
Hallewell RA, Mills R, Tekamp-Olson P, Blacher R, Rosenberg S, Ötting F, Masiarz FR, Scandella CJ (1987) Amino terminal acetylation of authentic human Cu, Zn superoxide dismutase produced in yeast. Biotechnology 5:363–366
Krafchich B (1989) Lyme disease. Int J Dermatol 28:71–74
Loison G, Vidal A, Findeli A, Roitsch C, Balloul JM, Lemoine Y (1989) High level of expression of a protective antigen of schistosomes in Saccharomyces cerevisiae. Yeast 5:497–507
Malawista SE (1989) Pathogenesis of Lyme disease. Rheumatol Int 9:233–235
Mendoza-Vega O, Hebert C, Brown SW (1994a) Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design. J Biotechnol 32:249–259
Mendoza-Vega O, Sabatié J, Brown SW (1994b) Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 15:369–410
Metzger J, Jung G, Bessler WG, Hoffmann P, Strecker M, Lieberknecht A, Schmidt U (1991) Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy) h heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages, and adjuvanticity in vivo and in vitro. J Med Chem 34:1969–1974
Schüller HJ, Entian KD (1988) Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product. Gene 67:247–257
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mendoza-Vega, O., Keppi, E., Bouchon, B. et al. Recombinant outer-surface protein A (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi: high production levels in Saccharomyces cerevisiae yeast cultures. Appl Microbiol Biotechnol 44, 624–628 (1996). https://doi.org/10.1007/BF00172495
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00172495