PRODUCTION OF POLYOMAVIRUS-LIKE PARTICLES IN A Klgal80 KNOCKOUT STRAIN OF THE YEAST Kluyveromyces lactis

Abstract

VP1, the major coat protein of polyomavirus, assembles intracellularly to virus-like particles if expressed in eukaryotes. Here, the nonconventional yeast Kluyveromyces lactis was used for production of virus-like particles of murine polyomavirus. The heterologous gene of VP1 was integrated in the LAC4 locus of the GAL/LAC genes. Consequently the expression of VP1 is regulated by the interplay of the activator KlGal4p and inhibitor KlGal80p. This cloning strategy couples the production of VP1 to that of the enzyme β-galactosidase, allowing a fast alternative for monitoring the course of recombinant protein production by measuring the β-galactosidase activity. A Klgal80 knockout strain was generated for a constitutive expression of VP1 and a continuous VLP production. High-cell-density fermentation showed that (1) Kluyveromyces lactis is generally suitable for VLP production and (2) the Klgal80 knockout strain produces higher amounts of recombinant VP1.

Furthermore, VLPs could be purified chromatographically to 87% (w/w) of total protein, and showed a homogeneous species of 45-nm particles and a high resistance against proteolysis compared to conventional in vitro assembled VLPs. This demonstrates the superior stability of virus-like particles produced in yeast. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Keywords:

• fermentation
• KLGAl80
• Kluyveromyces lactis
• polyoma VP1
• recombinant protein expression
• VLP
• yeast

ACKNOWLEDGMENTS

We thank the DFG/GRK 1026 for funding the research project. Furthermore, we thank Gerd Hause (Martin-Luther-Universität Halle-Wittenberg, Microscopy Unit, Halle, Germany) for electron microscopy of purified VLPs. We thank Hannes Grabner (Martin-Luther-Universität Halle-Wittenberg, inno profile, Halle, Germany) for assistance with substrate measurements with the plate reader. We also thank Holger Herrmann (Martin-Luther-Universität Halle-Wittenberg, inno profile, Halle, Germany) for data processing and density gradient simulation. Further, we thank Katja Diekow for the cross-breeding experiments. We acknowledge Thomas Klose (Purdue University, Rossmann Lab, West Lafayette, IN) for the purified bacteria VP1. We further acknowledge Karin Sorge (Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry and Biotechnology) for Southern blot analysis.

Notes

Two (1,2) cultivations of Kluyveromyces lactis with Klgal80p knock out and one (3) cultivation with functional Klgal80p were run for 96 h under identical conditions on supplemented rich medium with substrate (galactose) limitation. For run 3 product VP1 could not be determined quantitatively due to too low production yields.