Design of vectors for efficient integration and transformation in Hansenula polymorpha.
Four vectors were constructed for high-level expression of heterologous proteins with high copy number and mitotic stability in Hansenula polymorpha. All of them contained the conserved H. polymorpha-derived ribosomal DNA (rDNA) sequence for targeting and the geneticin (G418) resistance gene as a selection marker. A strong inducible promoter, formate dehydrogenase (FMD) promoter from H. polymorpha, was used to drive the expression of heterologous genes; the formate dehydrogenase terminator of H. polymorpha was used as the transcription termination region. A modified green fluorescent protein (mGFP) and firefly luciferase protein (Luc) were used as the marker to evaluate the efficacy of these vectors. Using Southern blotting analysis, 2-30 copies of these vectors were integrated into rDNA loci. These results demonstrated that all the four vectors could be used as candidates for expression of desired proteins in H. polymorpha.
Liu
Yueyong
Y
Molecular Microbiological Center, Institute of Microbiology, Chinese Academy of Sciences, 13# Zhongguancun Beiyitiao, 100080, Beijing, PR China.
Li
Yong
Y
Liu
Liyue
L
Hu
Xuebo
X
Qiu
Bingsheng
B
D001483:Base Sequence; D004275:DNA, Ribosomal; D015966:Gene Expression Regulation, Fungal; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D049409:Luciferases, Firefly / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic
D004275:DNA, Ribosomal; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly