CAJ | 학술논문

巴斯德毕赤氏酵母表达系统已被广泛用于生产外源蛋白的寄主菌。利用该系统将外源基因整合交换到染色体上时，AOX1基因被破坏，使表达菌的甲醇利用缓慢，给发酵生产造成一定影响。在不改变现有表达系统前提下，从AOX1功能缺陷菌株分离出Mut+自发突变体。通过突变体在甲醇培养基中生长曲线的测定，HSA表达产物的聚丙烯酰胺凝胶电泳检测，证明突变体的甲醇利用能力和蛋白表达比原始菌株大大提高；突变体AOX2基因上游区域经PCR扩增进行全序列测定，结果表明在PAOX2阻遏蛋白结合区域-255和-529两个碱基发生改变，该突变改善了AOX1基因缺陷功能，有利于外源基因的高表达。
파사덕필적씨효모표체계통이피엄범용우생산외원단백적기주균。이용해계통장외원기인정합교환도염색체상시，AOX1기인피파배，사표체균적갑순이용완만，급발효생산조성일정영향。재불개변현유표체계통전제하，종AOX1공능결함균주분리출Mut+자발돌변체。통과돌변체재갑순배양기중생장곡선적측정，HSA표체산물적취병희선알응효전영검측，증명돌변체적갑순이용능력화단백표체비원시균주대대제고；돌변체AOX2기인상유구역경PCR확증진행전서렬측정，결과표명재PAOX2조알단백결합구역-255화-529량개감기발생개변，해돌변개선료AOX1기인결함공능，유리우외원기인적고표체。
Spontaneous Mut+ mutants of P. Pastoris AOX1-defective expression strain have been isolated, they were identified as phenotypically utilized methanol to grow as wild type. The results obtained from measuring growth curve when cultivated in medium in which methanol as a sole carbon source and detecting HSA protein on SDS-PAGE confirmed that the mutants have increased ability to utilize methanol and express foreign HSA gene product. The promoter region of AOX2 gene from the mutants has been cloned by PCR amplification, and the DNA fragment is 1022bp in size. Sequencinganalysisshowedthattherearetwopointmutationsatpositions of -529 and -255 from the translation initiation codon respectively. The mutations improved AOX-1 defective function and facilitate the foreign gene for higher expression.