Subjects: Biology >> Bioengineering submitted time 2018-01-16 Cooperative journals: 《中国生物工程杂志》
Abstract：SUMO蛋白酶（Ulp1）是切割小分子泛素修饰（SUMO）融合蛋白获得天然N端靶蛋白的一种工具酶，具有酶切效率高，特异性好等优点。但现有市售SUMO蛋白酶Ulp1价格昂贵，操作复杂，限制了SUMO融合体系的运用。利用基因工程技术，合成基因ulp1（Leu403-Lys621），并在N端和C端加入多聚组氨酸标签（His6），构建重组表达载体psvT7-ulp1，将重组质粒转入大肠杆菌BL21(DE3)和BL21 trxB(DE3)中。经过高通量筛选技术快速确定最优的表达条件为采用BL21(DE3)作为表达宿主，转接后7 h加入IPTG，IPTG的终浓度为0.1 mmol/L，诱导时间为16 h，最终蛋白表达量占菌体总蛋白的34.5%，重组蛋白Ulp1的表达量为190mg/L，通过Ni-NTA一步纯化即可得到纯度95%以上的Ulp1。通过酶切反应，测定酶活为5.19 U/μl，比酶活为5.23×104 U/mg，是先前报道比酶活的1.87倍，通过酶活动力学分析，Ulp1的表观米氏常数 Km=0.359 g/L，Vm=5.10μg/(mL·min)。将SUMO融合表达体系用于单链抗体（Single-Chain Antibody Fragment，scFv）的表达，得到可溶的SUMO-scFv融合蛋白，使用表达的Ulp1进行酶切并纯化，获得纯度高于90%且N端不含多余氨基酸的scFv,操作步骤简单，显著改善了scFv在大肠杆菌中难于高效可溶性表达纯化的现状。
Subjects: Biology >> Bioengineering submitted time 2017-07-24 Cooperative journals: 《中国生物工程杂志》
Abstract： Abstract: object Separation and identification glycerol transporter in pichia pastoris (P. pastoris) and studying its function. Methods: In this study, the candidate glycerol transporter (gt1, GeneID: 8197545) was found in NCBI database by bioinformatics, and transmembrance structure was predicted by DAS. Targeted gene fused with egfp was cloned into pPICZ B and pRS424 to study its location in cells; recombinant plasmid (gt1+ pRS424) was transformed into Schizosaccharomyces pombe (S. pombe) to study its function; In order to study the relationship between gt1 and aox1, gt1 was deleted by homologous recombination, and enzyme activity of AOX1 in wild and mutant were detected. Results: According to results of bioinformatics, it was found that GT1 share the same transmembrance regions with sugar transporter 1( stl1) which has been identified as a glycerol transporter in Saccharomuces cerevisiae (S. cerevisiae), results of subcellular localization showed that GT1 located on membrance, S. pombe transformed into gt1 could growth on glycerol medium, but wild type could not, which suggested that GT1 could absorbed glycerol from medium to support cells growth. Moreover, AOX1 achieved constitutive expression in Δgt1. Conclusion: glycerol transporter (gt1) in P. pastoris was separated and identified, and its function in glycerol repression was preliminarily clarified.
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