Effect Of ATG Initiation Codon Context Motifs On The Expression Efficiency Of EGFP Under The AOX1 Promoter In Pichia Pastoris

P.pastoris, a kind of methanol nutrition yeast, has become the most important tools of genetic engineering production of heterologous protein,compared with other expression system, it has many advantages, for Escherichia coli expression system,which has the advantages of simple operation, low production cost, exogenous protein high yield, at the same time, also has the protein post-translational modification effect;for Saccharomyces cerevisiae, with high expression plasmid stability, expression strain is stable and secretion efficiency higher; for the mammalian expression system,it has the characteristics of simple operation, low culture medium, and not easy to be polluted in the process of production; The system has the advantage of significant cost reduction for the insect cell expression system.Gene expression regulation plays an important role in the process of protein expression, gene expression is divided into DNA transcription into RNA and RNA translated into protein. Transcription is the initial stage of gene expression, and regulation in this stage is the most economical one. In eukaryotes, gene regulation is mainly carried out at the transcriptional level. Through the interaction of cis acting elements and trans acting factors, the transcriptional regulation of eukaryotes is realized. Gene regulation of organism mainly occurred in these suitable on the cis acting elements, such as start codon, GC box, CCAAT box, enhancer, silencers,repressor, recently found that start codon ATG bases around affect gene transcription efficiency, affect the final protein production.A large number of experimental results show that the sequence around the start codon ATG has different effects on the expression of protein. Tatematsu, K., al. et published in 2014, it studied the sequence around initiation codon ATG of the silkworm on the expression of the recombinant protein. The article compared 50 kindsof silkworm gene, found AAAAATCAAAATGG sequence affects the efficiency of transcription, after they use EGFP and luciferase as reporter genes, construct 8 kinds of plasmid composed of different sequences, the sequence length is 14 bases,including ATG initiation codon, found that the sequence has a significant impact on gene transcription and protein production, and it has tissue specificity. Dai, C., et al.In 2007 published the article reports: They insert the potassium channel scorpion toxin gene into the p EGFP-N1 eukaryotic expression vector, and before the fusion protein of the initiation codon ATG to insert the Kozak sequence GCCACC, found that the inserted Kozak sequence GCCACC makes fusion protein expression increased greatly, but there is no expression of the not inserted Kozak sequence. Sano ki et al in 2002 article reported,they insert the 21 amino acids before ATG into the lobster’s original myosin c DNA,which was in the front of the fluorescein gene as well as the start codon ATG of the lobster, was found to significantly improve the expression of the two proteins. These reports show that the sequence around ATG has a great influence on the expression of genes, but there is no study on the effect of pichia pastoris ATG on the expression of AOX1 in the sequence of promoter.This experiment adopts the EGFP as a reporter gene, construct the recombinant plasmid p PICZαA-EGFP, according to α signal peptide sequence and EGFP sequence,design a pair of primers, Pst I restriction enzyme cleavage site was introduced in the upstream primer, and around the initiation codon ATG insert into six mutation bases,in E.coli, the recombinant plasmid was amplified and then transformed into Pichia pastoris, the expression amount of EGFP was measured.nduced transformants expressed and screened positive transformants, By repeated sequencing, 77 positive clones were obtained in the front of ATG; 48 positive clones were obtained in the back of ATG. The sequence of the mutated bases were recorded, The sequence of the mutated bases were recorded, and then we used RT-q PCR method to measure the m RNA levels of these clones, and comprehensively analyze the influence of sequence around ATG on the transcription and translation of EGFP gene.The experimental results show that:(1) In front of ATG into intert six bases, a significant increase in EGFP expression(fluorescence value changes rate is higherthan 100%) of the sequence is CMSYMM; the moderate degree of enhancement of EGFP expression(the rate of fluorescence change rate in 50-100%) of the sequence is RHSDYC; the increase of EGFP expression was not affected by the sequence(the rate of change of fluorescence value in 0-50%) is CMBNCB; The sequence of decreasing EGFP expression(negative value of fluorescence change rate) was KWBKTN. The expression level of m RNA was basically consistent with the change of EGFP fluorescence intensity of the sequence of the inserted sequence in front of the ATG, it shows that the sequence inserted in front of the ATG is affected by the transcription of EGFP, the expression of m RNA was changed, which affected the translation of protein.(2) In back of ATG into intert six bases, significantly enhanced EGFP expression(fluorescence change rate is higher than 100%) of the sequence is BHYRAS; the moderate degree of enhancement of EGFP expression(the rate of fluorescence change rate in 50-100%) of the sequence is ASBRWY,the enhancement effect is weak(the fluorescence value change rate is in 0-50%) of the EGFP expression quantity of the sequence is NHCVND; The sequence of decreasing EGFP expression(negative value of fluorescence change rate) was WARRBV. The expression level of m RNA was basically consistent with the change of EGFP fluorescence intensity of the sequence of the inserted sequence in the back of the ATG,it shows that the sequence inserted in the back of the ATG is affected by the transcription of EGFP, the expression of m RNA was changed, which affected the final yield of protein.