CAJ | 학술논문

目的：通过过表达N－糖基转移酶，构建1株糖基化修饰效率更高的糖基工程酵母。方法利用尿嘧啶（URA3）营养缺陷型筛选标记，在糖基工程酵母4－32中转入醇氧化酶1（alcohol oxidase 1，AOX1）启动子控制的利士曼原虫N－糖基转移酶星状孢子素和温度敏感性酶3（staurosporine and temperature sensitivity 3，STT3）D亚基，通过SDS－PAGE、Western印迹和肽N－糖苷酶F（peptide－N－asparigineamidase F，PNGase F）酶切等方法，分析4－32－STT3D表达的抗人表皮生长因子受体2（HER2）抗体（anti－human epidermal growth factor receptor 2）和粒细胞－巨噬细胞集落刺激因子（granulocyte－macrophage colony－stimulating factor，GM－CSF）N－糖基化程度，并测定STT3D转入和诱导表达对酵母生长情况的影响。结果 SDS－PAGE结果显示，4－32－HL菌表达的抗HER2抗体除有一条相对分子质量约55×103的重链主带外，还有一条约50×103的未糖基化条带。4－32－HL－STT3D菌表达为均一的55×103条带，无50×103条带。 PNGase F酶切后，上述重链呈50×103的均一条带。 Western印迹证明以上所有条带均为抗体成分。以GM－CSF作为报告蛋白验证STT3D的作用，结果显示，4－32－GM－CSF菌表达的GM－CSF为22×103和20×103的两条带，而4－32－GM－CSF－STT3D表达则为均一的22×103条带。 PNGase F酶切4－32－GM－CSF和4－32－GM－CSF－STT3D菌表达的GM－CSF后，GM－CSF呈18×103的均一条带。分别测定STT3D外源基因转入和诱导表达时对酵母生长情况的影响，统计学分析显示，STT3D转入不诱导对酵母生长影响不显著，STT3D诱导表达对酵母生长影响极显著。结论过表达N－糖基转移酶的糖基工程酵母对靶标蛋白具有更高的N－糖基化修饰效率。
목적：통과과표체N－당기전이매，구건1주당기화수식효솔경고적당기공정효모。방법이용뇨밀정（URA3）영양결함형사선표기，재당기공정효모4－32중전입순양화매1（alcohol oxidase 1，AOX1）계동자공제적리사만원충N－당기전이매성상포자소화온도민감성매3（staurosporine and temperature sensitivity 3，STT3）D아기，통과SDS－PAGE、Western인적화태N－당감매F（peptide－N－asparigineamidase F，PNGase F）매절등방법，분석4－32－STT3D표체적항인표피생장인자수체2（HER2）항체（anti－human epidermal growth factor receptor 2）화립세포－거서세포집락자격인자（granulocyte－macrophage colony－stimulating factor，GM－CSF）N－당기화정도，병측정STT3D전입화유도표체대효모생장정황적영향。결과 SDS－PAGE결과현시，4－32－HL균표체적항HER2항체제유일조상대분자질량약55×103적중련주대외，환유일조약50×103적미당기화조대。4－32－HL－STT3D균표체위균일적55×103조대，무50×103조대。 PNGase F매절후，상술중련정50×103적균일조대。 Western인적증명이상소유조대균위항체성분。이GM－CSF작위보고단백험증STT3D적작용，결과현시，4－32－GM－CSF균표체적GM－CSF위22×103화20×103적량조대，이4－32－GM－CSF－STT3D표체칙위균일적22×103조대。 PNGase F매절4－32－GM－CSF화4－32－GM－CSF－STT3D균표체적GM－CSF후，GM－CSF정18×103적균일조대。분별측정STT3D외원기인전입화유도표체시대효모생장정황적영향，통계학분석현시，STT3D전입불유도대효모생장영향불현저，STT3D유도표체대효모생장영향겁현저。결론과표체N－당기전이매적당기공정효모대파표단백구유경고적N－당기화수식효솔。
Objective To obtain a strain of glycoengineering yeast with higher N-glycosylation efficiency by overexpressing N-glycosyltransferase.Methods Through the selecting marker URA3 gene, a new glycoengineering yeast strain named 4-32-STT3D was constructed, which could overexpress the Leishmania major N-glycosyltransferase staurosporine and temperature sensitivity3 D subunit(STT3D) under the control of an inducible alcohol oxidase 1(AOX1) promoter.We analyzed the N-glycosylation status of anti-human epidermal growth factor receptor 2 ( HER2 ) antibody and granulocyte macrophage colony stimulating factor (GM-CSF) expressed in 4-32-STT3D using SDS-PAGE,Western blotting and peptide-N-asparigineamidase F(PNGase F).Finally the effect of STT3D on the growth rate of glycoengineering yeast was detected.Results SDS-PAGE showed that anti-HER2 antibody expressed in 4-32-HL had two components:the first one with a relative molecular mass 55 ×103 was glycosylated,while the second one with 50 ×103 was non-glycosylated,but anti-HER2 antibody expressed in 4-32-HL-STT3D had the component of 55 ×103 only without any non-glycosylated 50 ×103 .The above components became 50 ×103 with the digestion of PNGaseF.All of them proved to be antibodies by Western blotting.As a report protein,GM-CSF expressed in 4-32-GM-CSF had two components: 22 ×103 and 20 ×103, while in 4-32-GM-CSF-STT3D there was only one with 22 ×103 .All these components became 18 ×103 with the digestion of PNGase F.Statistical analysis showed that without induction,STT3D had no effect on the growth rate of glycoengineering yeast, while great effect was observed when STT3D was induced.Conclusion Glycoengineering yeast with the overexpression of N-glycosyltransferase has higher N-glycosylation efficiency.