SUMO蛋白酶Ulp1的高效表达纯化并通过His-SUMO标签制备scFv

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(His_6),构建重组表达载体psv T7-ulp1,将重组质粒转入大肠杆菌BL21(DE3)和BL21 trx B(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K_m=0.359g/L,V_m=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。     

融合蛋白GST-Ulp1p在大肠杆菌中的高效可溶性表达及其活性鉴定

利用基因工程技术,体外重组小分子类泛素修饰蛋白酶1(Ulp1)的活性片段,获得高表达、高特异性重组蛋白酶。从酿酒酵母Saccharomyces cerevisia中提取Ulp1编码第403到621个氨基酸残基之间的DNA片段(Ulp1p),在其C端加入6×His并连接到大肠杆菌表达载体pGEX中,构建重组表达质粒pGEX-Ulp1p-his6。将重组质粒转化至大肠杆菌Rosetta(DE3)中,氨苄青霉素抗性筛选转化子。表达、纯化后,以SUMO融合蛋白检测其活性。经过优化,该蛋白可溶性表达,表达量占菌体总蛋白的40.12%。可通过谷胱甘肽琼脂糖凝胶柱或Ni-NTA凝胶亲和层析纯化得到纯度98%的蛋白。经酶切分析,比活力为1.375×104U/mg。融合蛋白GST-Ulp1p-His6无需切除谷胱甘肽S-转移酶(GST)标签,具有很高的活性,制备简易;6×His标签,有利于底物蛋白切割后纯化,减少蛋白损失。本研究为制备高活力的SUMO蛋白酶提供了一个新方法。     

构建一种精确表达小分子蛋白的技术平台

目的:克服原核表达的小分子蛋白难以去除附加氨基酸的问题,为基因工程精确表达小分子蛋白提供一种简便有效的解决方案.方法:以73个氨基酸的小分子蛋白(黑色素瘤生长激活因子CXCL1的功能区)为例,用PCR方法扩增了基因,TA克隆到载体PET SUMO.测序验证后的重组质粒转化至表达茵BL21(DE3)中诱导表达,融合蛋白用基质辅助解吸电离飞行时间质谱仪进行串联质谱鉴定(MALDI-TOF-MS/MS).通过特异性的SUMO蛋白酶酶解载体SUMO蛋白,利用SUMO蛋白和其蛋白酶带6x His标签的性质,经镍螯合柱亲和层析将二者去除,以Western blotting证明目的小分子蛋白的分离.结果:①经测序,重组质粒无突变,TA克隆方向正确,重组载体构建成功.②MALDI-TOF-MS/MS证明融合蛋白由SUMO蛋白和CXCLl蛋白组成,除去SUMO蛋白后的表达终产物经Westem blotting鉴定,与其相应的抗体有特异性结合,证明分离得到的小分子蛋白即为目的蛋白.结论:运用这一技术可将载体蛋白完全去除从而达到精确表达小分子蛋白的目的,在分子生物学的实验研究中有很好的应用前景.     

高效可溶性重组蛋白表达载体的构建

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。     

小分子泛素相关修饰物SUMO及其融合表达

小分子泛素相关修饰物(small ubiquitin-related modifier,SUMO)是一类具有高度保守序列的低分子量蛋白.在蛋白质的融合表达中,SUMO作为融合标签得到了广泛应用.与其他传统融合标签如谷胱甘肽-S-转移酶、二硫键形成蛋白A、硫氧还蛋白等相比,SUMO具有防止蛋白降解、促进蛋白折叠、能被SUMO蛋白酶专一性识别切割并在目的蛋白上不会留下氨基酸残基等优点.本文综述SUMO融合蛋白及其配套的SUMO蛋白酶在融合表达中的优势和应用.     

重组人神经生长因子β亚基的原核优势表达及纯化

目的:在原核系统内表达SUMO融合重组人神经生长因子β亚基(h NGF-β),并对其纯化。方法:将5'端引入了羟胺切割位点的h NGF-β基因克隆到表达载体p ET-SUMO中,IPTG诱导表达后,对表达的融合蛋白SUMO-h NGF-β包涵体进行亲和纯化,然后在变性条件下加入羟胺进行裂解,利用SUMO分子伴侣的功能与h NGF-β进行共复性,最后利用阳离子交换层析纯化获得重组h NGF-β。结果:融合蛋白SUMO-h NGF-β相对分子质量为39×103,表达量占细菌总蛋白的35%;在变性条件下经羟胺切割、共复性和阳离子纯化后,相对分子质量为14×103的重组h NGF-β复性率和纯度分别为30%和80%以上,Western印迹显示其具有良好的抗原特性。结论:h NGF-β与SUMO融合可以在原核表达系统中实现优势表达。     

SUMO蛋白酶活性片段的表达、纯化及活性测定

利用PCR技术人工合成编码酿酒酵母泛素样特异性蛋白酶1 (Ubiquitin-like specific protease 1,Ulp1)第403到621个氨基酸残基之间的DNA片段Ulp1p,并连接到大肠杆菌表达载体pET-3c中,构建出重组表达质粒pET-Ulp1p。将重组质粒转化至大肠杆菌BL21(DE3)中,氨苄青霉素抗性筛选转化子。经IPTG诱导4h后, SDS-PAGE结果显示,Ulp1p为可溶性表达,表达量占菌体总蛋白的50.8%。通过Ni-NTA凝胶亲和层析和G-25凝胶层析联用可以获得纯度大于95%的Ulp1p。Western-blotting分析表明,Ulp1p能与6xHis抗体产生免疫反应。以重组蛋白SUMO-hEGF(人表皮生长因子)和GST-SUMO-MT(金属硫蛋白)为底物进行酶切分析,结果显示,Ulp1p能特异性水解这两种SUMO融合蛋白,其比活为1.386 x104U/mg。     

胸腺肽α1-Spl DnaX内含肽融合蛋白的切割动力学研究

目的:研究胸腺肽α1(Tα1)与Spl DnaX内含肽融合蛋白AS的体外切割动力学。方法:构建Tα1和SplDnaX内含肽的融合表达载体pET-AS,转化大肠杆菌BL21(DE3),经乳糖诱导获得可溶性表达的融合蛋白AS,用镍亲和层析纯化该蛋白;综合评价温度、β-巯基乙醇(β-ME)浓度和诱导切割时间对Spl DnaX内含肽介导融合蛋白AS自切割释放Tα1的影响。结果:在大肠杆菌中诱导表达了融合蛋白AS,经镍柱纯化获得该蛋白;随着温度升高和β-ME浓度增加,诱导切割时间延长,Spl DnaX内含肽介导的切割率逐渐增大;最终采用300 mmol/Lβ-ME切割24 h,融合蛋白的硫解切割率大于90%。结论:通过对Spl DnaX内含肽的诱导切割条件进行摸索,确定了最适宜的切割条件,为利用该方法制备Tα1和其他小分子多肽提供了技术基础。     

高效表达可溶性重组蛋白表达载体——pHisSUMO

目的:构建含有IL-1基因的原核表达质粒,并对其原核表达情况进行检测,验 证pHisSUMO表达载体的高效可溶性表达.方法:以质粒pMD18-T-IL-1为模板,利用PCR获得I L-1基因克隆并将其与表达载体pET、pTYB、pHisSUMO连接,重组质粒经鉴定后转化到大肠杆 菌DH5α中,并检测其蛋白表达情况.结果:仅在pHisSUMO表达系统获得了IL-1融合蛋白的 高效可溶性表达.利用Ni-NTA纯化后的融合蛋白经SUMO蛋白酶Ⅰ切割,获得了纯度较高的成熟 蛋白且不残留任何氨基酸残基.结论:实验证明pHisSUMO表达系统有助于增加外源蛋白可溶性和表达量.     

鲮鱼皮胶原肽的制备及其抗氧化活性的检测

为了以细菌胞外蛋白酶酶解低值蛋白资源制备抗氧化活性肽以及挖掘新型蛋白酶,采用液体发酵培养的方法对假交替单胞菌Pseudoalteromonas sp.SHK1-2进行发酵产酶,获得胞外蛋白酶粗酶液用于酶解热水法提取的鲮鱼胶原蛋白,通过超滤、sephadex LH-20分子筛层析获得具有DPPH自由基清除能力(35.6%±7%)、氧自由基清除能力(Oxygen radical absorbance capacity,ORAC)及DNA氧化损伤抑制活性的小分子肽,液相色谱质谱联用鉴定该活性肽分子量为776.2 Da,预测氨基酸序列为Thr-Ala-Gly-His-Pro-Gly-Thr-His。ORAC检测验证了人工合成的多肽同样具有抗氧化活性。研究表明细菌胞外蛋白酶在低值资源高值化中具有一定的应用前景,对于新型蛋白酶及其应用的挖掘具有一定的参考意义。     

Biodiversity in aerobic granule membrane bioreactor at high organic loading rates

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.     

Production of Bioactive Human Beta-Defensin-4 in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner

The human beta defensins-4 (hBD4) exhibit a broad range of antimicrobial properties and are thought to be ideal therapeutic agents because of their potential ability to circumvent the problems of acquired resistance often observed with other antimicrobial therapies. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide hBD4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 637 mg protein of interest was obtained per liter of fermentation culture. After the SUMO-hBD4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 166 mg recombinant hBD4 was obtained from 1 L fermentation culture with no less than 96% purity and the recombinant hBD4 had similar antimicrobial properties to the synthetic hBD4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.     

Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC₅₀ of 1.77 ± 0.53 μg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in this study allows convenient high yield and easy purification of small recombinant peptides with native sequences. Z. Sun and Z. Xia contribute equally to the work.     

Recombinant production of self-assembling SZ-structured peptides using SUMO as a fusion partner

ABSTRACT: BACKGROUND: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P11-family of peptides includes, P11-4 (QQRFEWEFEQQ) and the complementary peptides P11-13 (EQEFEWEFEQE) and P11-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P11-4) or when mixed (P11-13 and P11-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage. RESULTS: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy. CONCLUSIONS: The fusion proteins were produced in high yields and the beta-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.     

Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.     

Production of Bioactive γ-Glutamyl Transpeptidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner and Application of the Recombinant Enzyme to <Emphasis Type="SmallCaps">l</Emphasis>-Theanine Synthesis

The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine.     

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.     

High Efficient Expression of Bioactive Human BMP-14 in <Emphasis Type="Italic">E. coli</Emphasis> Using SUMO Fusion Partner

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-β superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of human BMP-14. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography. After the SUMO-BMP14 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 45 mg recombinant hBMP-14 was obtained from 1 litre bacterial culture with no less than 95% purity. The purified hBMP-14 dimer was over 90% purity and could induce the expression of alkaline phosphatase activity in C2C12 cells in a dose-dependent manner. Thus the SUMO-mediated peptide expression and purification system potentially could be employed for the production of other homodimeric proteins.     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.