Construction And Rapid Screening Of Pichia Pastoris Over-expressing Recombinant Protein

As a mature protein expression system,Pichia pastoris system not only has the characteristics of simple operation and fast growth,but also has the post-translational modification processing system of eukaryotic cells,and has low nutritional requirements.It can use cheap medium,and is suitable for high-density fermentation.It is widely used in recombinant protein expression.Ribosomal rDNA integration is a rapid and efficient method for the construction of multi copy strains.A series of strains with different copy numbers can be obtained quickly based on resistance stress and screening by connecting the non essential highly repetitive gene fragment from Pichia pastoris rDNA,non-transcribed intergenic spacer(NTS),at both ends of the target gene.In addition,the recombinant strains were cultured in liquid medium based on the method of posttransformational vector amplification in liquid medium(liquid ptva).The strains with different copy numbers were selected by using antibiotic pressure.Combined with the adaptive evolution system of microfluidic microbial culture and flow cytometry,the fluorescent labeled high-yield recombinant foreign protein expression strains were screened,it provides an important basis for further rapid screening of Pichia pastoris strains with high expression of recombinant protein.The main research contents and results are as follows.(1)GS115-E strain was constructed by fusion expression of EGFP and Sec63 in Pichia pastoris,and it was confirmed that it had no effect on cell growth and production of recombinant protein.Phytase and xylanase were expressed in GS115-E,the correlation between different fluorescence values and phytase expression or xylanase expression was analyzed in shake flask culture.The results showed that there was a general and good linear correlation between the expression level of EGFP fused with Sec63 and the expression level of phytase recombinant protein(0.8<|R|<1).Furthermore,flow cytometry was used to select the low fluorescence and high fluorescence cell groups from the mixed bacterial groups with different expression levels of phytase.The expression level of phytase in the latter was 4.09 times higher than that in the former after 120 h in shake flask culture.By analyzing the EGFP fluorescence value of yeast cells instead of detecting the expression and activity of recombinant protein,the convenience and versatility of the method were greatly improved,which provided a rapid screening method for Pichia pastoris strains with high expression of recombinant protein.(2)On the basis of constructing a rapid screening method for Pichia pastoris strains with high expression of recombinant protein,the high expression strains were constructed and screened by inserting the target gene in the non-transcribed intergenic spacer,using Liquid PTVA adaptive evolutionary culture method and flow cytometry based on droplet microfluidic technology.The antibiotic zeocin was gradually promoted in the culture drops of MMC system,so that the constructed GS115/phy/NTS strain could carry out adaptive evolution.At the concentration of zeocin was 100 ?g/m L,a total of 9 strains with phytase recombinant expression level 3 times or more than that before evolution were screened(among them,4 of the 6 selected strains expressed phytase activity 3 times or more than that before evolution,and the highest was 3.80 times;after 96 well plate culture and shake flask culture,the enzyme activity of 5 of the 6 selected strains was 3 times or more than that before evolution,and the highest was 3.95times).The concentration of zeocin was 200 ?g/m L,the phytase expression level of five strains was 3 times or more than that before evolution,and the highest was 4.33 times of that before evolution.The concentration of zeocin was 300 ?g/m L,the results showed that more than 60%of the selected high fluorescence strains had 4-fold or more expression of phytase,and the highest expression level of phytase was 3.50 times of that before evolution.The construction and rapid screening method of Pichia pastoris strain with high expression of recombinant protein was established.