The Expression Of Human Insulin Precusor In Pichia Pastoris

As specific drugs for diabetes treatment, the demand for insulin has been embarking on asteep rising trend in recent years. Most of active human insulin is processed from humaninsulin precusor by genetic engineering now. And human insulin precusor is generallyexpressed by Eco.li and yeast expression system. Owing to more advantage for expressingheterologous protein, human insulin precusor is expected to achieve high expression by Pichapastoris. In this paper, strategies to increase human insulin precusor production wereresearched on the basis of Picha pastoris X-33host, including increasing target gene copynumber, decreasing target protein degradation, optimizing high-density fermentationtechnology primarily.1First, the method of increasing target gene copy number was used. For this, the gene ofhuman insulin precursor (IP) was synthesized and ligated with pPICZα. Then the recombinantexpression vector pPICZα-IP was electronicly transformed into P. pastoris X-33. The mutantstrains B4and S6were screened on the YPDS plate containing100μg/mL zeocin, the productfor human insulin precursor was confirmed by flask culture, Tricine-SDS-PAGE and massspectrometry. With B4and S6as start strains, we repeatedly transformed them with SacIlinearized pPICZα-IP and screened a mutant strain2B4(with copy number7) on the1000μg/mL zeocin plate. Compared with the control strain B7(with copy number1.4), the humaninsulin precusor yield of2B4strain was2fold higher than that of B7and the cell growth wasnot inhibited. The result that target gene transcription level of2B4was2fold higher than thatof B7was discovered by real-time quantification PCR test. In conclusion, the strategy of thecombination of resistance screening and repeatedly electroporation is efficient to increase thecopy number of target gene, then to facilitate higher trancription level, resulted in higher yieldof objective recombinant product ultimately.2Upon the above study, the phenomenon that insulin precusor yield rapidly decreasedduring methanol induced96h and120h in the high-density fermentation was discovered. Invitro, protease inhibitor mixture was added into2B4strain broth and supernate at methanolinduced96h and120h. Compared with the negative control, addition of protease inhibitormixture was effective to slow down target protein degradation in the supernate, but ineffectivefor broth. More protease have proven existed in the broth than supernate, so as to be difficultto prevent degradation. As a result, yield decreasing was caused by a large number of proteasereleasing. By adding specific protease inhibitor into2B4strain broth at methanol induced96h, the result that Pepstatin A was useful to slow down target protein degradation was discovered, demonstrating that Pepstatin A was the main reason to cause insulin precusordegradation. So host yeast SMD1168lacking Pepstatin A activity was chosen to construct anew recombinant yeast, named HR9. After cultivating in5L fermentor, human insulinprecusor degradation could be relieved, but the growth rate was distinctly slower than that of2B4strain. Finally, insulin precusor production had no further increase.3In the end, technology optimization of high-density was studied. Cultivatingtempreture was set30°C,25°C,22°C, respectively, and25°C was best for human insulinprecusor producing. Compared with that at30°C, the yield of human insulin precusor at25°Chave increased by11.3%when methanol induced for72h. Ajusting the methanolconcentration to1g/L,2g/L,4g/L on the methanol induced stage, respectively, and2g/Lwas best which have increased the highest human insulin precusor production by40.0%.Compared with the negative control, addition of1%tryptone when methanol induced for60hcould increase the yield by14.8%and the degradation rate of human insulin precusor havedecreased by77.5%during methanol induced96h to120h stage.