重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

Expression and Purification of Functional HuGALNT3 without the Transmembrane Domain(huGALNT3-sol) in Pichia pastoris

目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3(GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性。方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1 755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母GS115。通过MD平板和G418平板筛选出阳性高拷贝重组菌株。阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化。分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性。结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株。阳性表达菌株在BMMY培养基(pH 6.0)中20℃培养,经0.5%甲醇诱导表达96 h,摇瓶表达量可达5mg/L。SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式。活性检测显示表达的重组蛋白具有转糖基活性。结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础。

Objective：In order to research the bioactivity of GALNT3,the truncated part of GALNT3（huGALNT3-sol） which was deleted of the hydrophobic trans-membrane domain were obtained using Pichia pastoris expression system,and assayed the transferring GalNAc activity of recombinant huGALNT3-sol.Methods： The gene of human GALNT3-sol（1 755 bp）was amplified from pET15b/ GALNT3-sol and cloned into expression vector pPIC9k,and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation.The high copy recombinant strains with high-level huGALNT3-sol production were screened out by MD plate and G418.High level of huGALNT3-sol was obtained in BMMY medium the induction of methanol,and purified from the supernatant with Ni-NAT.The identity of the recombinant protein was confirmded by SDS-PAGE and then Western blotting analysis.HPLC and MALDI-TOF-MS analysis were used to identify the bioactivity of recombinant huGALNT3-sol.Results：The recombinant Pichia pastoris which could secretory express the human GALNT3-sol protein was constructed successfully.High level of huGALNT3-sol was obtained in BMMY medium（pH 6.0） after 96 hours induction of 20℃ and 0.5% methanol,with the highest yield of 5mg/L in shake flask culture.The identity of the recombinant protein was confirmed by Western blot analysis and the huGALNT3-sol expressed in Pichia pastoris is in higher molecular weight glycoforms.The activity assay showed the recombinant huGALNT3-sol has the activity of transferring GalNAc to Ser/Thr residues in peptide.Conclusion：The human GALNT3-sol,which has the activity of transferring GalNAc to Ser/Thr residues in peptide,was successfully expressed and purified in Pichia pastoris.It provides support for the further research and application of GALNT3.