Improvement Of Hansenula Polymorpha Expression Platform And Bovine FSH α,β Subunit Expression

The methylotrophic yeast, Hansenula polymorpha, has been successfully exploited as a cell factory for the large-scale production of heterologous genes in recent years.We use the Hansenula polymorpha technical platform,which is kept in our laboratory ,and modify it by adding 25SrDNA to it in order to increase copy numbers and then improve expression lever . A modified green fluorescent protein (mGFP5) and firefly luciferase protein (Luc)was used as the marker for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 and Luc thus expressed and retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. Additionally, 25SrDNA can increase copy numbers and improve expression lever of aim gene.We synthesized bovine Follicle Stimulating Hormone alpha subunit original gene segment according to FSH alpha subunit cDNA sequence Moreover we synthesized the other two different alpha gene segments according to Hansenula polymorpha codon prejudice and tRNA abundance we also synthesized bovine Follicle Stimulating Hormone beta subunit original gene according to the gene sequence from genebank Then we inserted those gene segments into Hansenula polymorpha expression vectors(pHFMD-Z-alpha-A) and induced after transformed into H. polymorpha.Using ELISA and Western blot to evaluate the experiment result ,it showed that FSH beta and FSH alpha subunit gene segment synthesized according to Hansenula polymorpha tRNA abundance expressed successfully in Hansenula polymorpha ,and the expression lever of alpha subunit is about 2mg/L .The chimera was purified through Ni+ resin column Moreover FSH alpha subunit sequence was inserted into pHFMD-Z-alpha-A-25s vector to compared with that in pHFMD--Z-alpha-A without 25s rDNA ,it indicated that 25s rDNA can improve the copy numbers and expression lever was improved greatly.What's more ,we used glycerin and methanol as different revulsant and compared their expression lever ,we found that methanol is much better than glycerin. It is the first time to express bovine FSH alpha subunit and beta subunit in Hansenula polymorpha and it has paved a good way for the whole glycoprotein Follicle Stimulating Hormone expressing in Hansenula polymorpha. We also found new ways to improve copy numbers.