Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis.
Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8-9% of the total cell protein.
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 79005, Ukraine.
Olena S
OS
Krasovska
Oleh V
OV
Stasyk
Andriy A
AA
Sibirny
D003001:Cloning, Molecular; D015870:Gene Expression; D005819:Genetic Markers; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D005819:Genetic Markers; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins