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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (3): 51-61    DOI: 10.13523/j.cb.20180307
技术与方法     
SUMO蛋白酶Ulp1的高效表达纯化并通过His-SUMO标签制备scFv *
李诗洁1,2,3,杨艳坤1,2,3,刘萌1,2,3,白仲虎1,2,3*(),金坚2,4*()
1 江南大学粮食发酵工艺与技术国家工程实验室 无锡 214122
2 江南大学工业生物技术教育部重点实验室 无锡 214122
3 江南大学糖化学与生物技术教育部重点实验室 无锡 214122
4 江南大学药学院 无锡 214122
Efficient Expression of SUMO Protease Ulp1 and Used to Express and Purified scFv by His-SUMO tag
Shi-jie LI1,2,3,Yan-kun YANG1,2,3,Meng LIU1,2,3,Zhong-hu BAI1,2,3*(),Jian JIN2,4*()
1 National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China
2 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School ofBiotechnology, Jiangnan University, Wuxi　214122, China
3 The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
4 School of Pharmacy,Jiangnan University, Wuxi 214122, China
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摘要：

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7h加入IPTG,IPTG的终浓度为0.1mmol/L,诱导时间为16h,最终蛋白质表达量占菌体总蛋白质量的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5.19U/μl,比酶活为5.23×10 4U/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=0.359g/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在大肠杆菌中难以高效可溶性表达纯化的现状。

关键词： Ulp1;  Ni-NTA;  SUMO;  scFv;  高效表达纯化    
Abstract:

SUMO protease (Ulp1) is an enzyme that cleaves small molecule ubiquitin-modified (SUMO) fusion protein into natural N-terminal protein,which has high efficiency and specificity.While the existing commercially available SUMO protease Ulp1 is expensive and complicated on operational level,limiting the wide application of SUMO fusion system. ulp1 gene (Leu403-Lys621) was synthesized,with the poly-histidine tag (His6) added to both the N-terminal and C-terminus of the SUMO protease(Ulp1).The recombinant expression vector psvT7-ulp1 was constructed before the recombinant plasmids was transformed into Escherichia coli BL21(DE3) and BL21 trxB(DE3) separately.The optimal expression conditions were as follows:Escherichia coli BL21 (DE3) was used as the expression host,and IPTG was added at 7h after the transfer.The final concentration of IPTG was 0.1mmol/L and the induction time was 16h.The final recombinant protein Ulp1 was 190mg/L accounted for 34.5% of the total protein in bacteria.Purified SUMO could be obtained through one-step Ni-NTA and the purity was higher than 95%.The enzymatic activity was 5.19U/μl with a specific enzyme activity of 5.23×10 4U/mg,which was 1.87 folds of the previously reported,according to enzyme kinetic analysis,the apparent Km of Ulp1 was 0.359g/L,and Vm was 5.10μg/(ml·min).The SUMO fusion expression system was then used to express the soluble SUMO-scFv fusion protein,with the expressed Ulp1 used for digestion and purification.The purity of scFv was higher than 90% and N-terminal natural scFv was obtained. A method was presented for preparation of Ulp1 with high enzyme activity and purity.The SUMO fusion expression system was successfully applied to the expression and purification of scFv with a simplified operation steps,which significantly improved the status of scFv soluble expression in Escherichia coli.

Key words: Ulp1    Ni-NTA    SUMO    scFv    High-performance purification
收稿日期: 2017-10-11 出版日期: 2018-04-04
ZTFLH:  Q814  
基金资助: 国家863计划(2015AA020802)
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李诗洁
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引用本文:

李诗洁,杨艳坤,刘萌,白仲虎,金坚. SUMO蛋白酶Ulp1的高效表达纯化并通过His-SUMO标签制备scFv *[J]. 中国生物工程杂志, 2018, 38(3): 51-61.

Shi-jie LI,Yan-kun YANG,Meng LIU,Zhong-hu BAI,Jian JIN. Efficient Expression of SUMO Protease Ulp1 and Used to Express and Purified scFv by His-SUMO tag. China Biotechnology, 2018, 38(3): 51-61.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180307    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/51

Primers names Primer sequences(5'-3')
His-sumo-F GGAATTCCATATGGGTCATCACCATCATCATCACG
sumo-R CGGACCGCTTTGCACCAGCT
scFv-F CAAATTCAGCTGGTGCAAAGCGG
scFv-R CCGCTCGAGTTATTTGATCTCCAGTTTGGTACCG
表1  本文所用的引物名称和序列
图1  His-sumo-scFv的合成流程
System(40μl) 1 2 3 4 5 6 7 8
SUMO-EGFP(μg) 15.57 15.57 15.57 15.57 15.57 15.57 15.57 15.57
Ulp1(ng) 0 49.555 99.11 128.843 148.665 168.487 188.309 198.22
10×SUMO Protease buffer(μl) 4 4 4 4 4 4 4 4
dd H2O 26 25.5 25 24.7 24.5 24.3 24.1 24
表2  SUMO蛋白酶酶切体系
system
(40μl)		 SUMO-EGFP
(μg)		 Ulp1
(ng)		 缓冲液
1 15.57 198.22 蛋白质纯化上样缓冲液(20mmol/L NaH2PO4·2H2O、50mmol/L NaCl,pH=8.0)
2 15.57 198.22 蛋白质纯化洗脱缓冲液(20mmol/L NaH2PO4·2H2O、50mmol/L NaCl、250mmol 咪唑pH=8.0)
3 15.57 198.22 高盐缓冲液(250mmol/L Tris-HCl,250mmol/L NaCl,pH=8.0)
4 15.57 198.22 PBS(pH=7.4)
5 15.57 198.22 10×SUMO Protease buffer(稀释10倍)
表3  SUMO蛋白酶酶切体系
图2  融合PCR扩增基因sumo-scFv和重组质粒psvT7-ulp1酶切验证
图3  SUMO蛋白酶的诱导表达
图4  Ulp1的Western blot鉴定
图5  SUMO蛋白酶的纯化及酶活鉴定
图6  SUMO-scFv在不同诱导条件下的表达情况及SUMO-scFv纯化
图7  SUMO-scFv蛋白酶切及scFv纯化
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