一种在大肠杆菌中促蛋白可溶性高效表达的ELP-SUMO新系统的建立及应用研究

【作者】 张杰；

【导师】 张双全；

【作者基本信息】 南京师范大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 重组蛋白广泛应用于生物学和生物药学的科学研究中。大肠杆菌表达系统是作为低成本、快速表达纯化重组蛋白进行研究的首选系统。但利用大肠杆菌表达制备异源蛋白时存在的问题是蛋白折叠不正确和蛋白不可溶。已有许多方法用来解决这些问题,如融合表达、共表达分子伴侣及改变启动子等。虽然改变表达的条件有时能解决一些问题,但到目前为止,最可行的方法还是融合标签融合表达来提高目的蛋白的可溶性；因此,蛋白融合表达被普遍用来提高目的蛋白的表达及可溶性。但到目前为止,没有一种融合标签能提高所有目的蛋白的表达量和可溶性,但其中有一些标签还是具有优势的,如SUMO标签已成为一个常用的标签,用来融合表达那些难可溶表达的蛋白。本论文主要着重于建立一种在大肠杆菌中促蛋白可溶性高效表达的新系统,将其应用表达一些在大肠杆菌以不可溶形式表达的蛋白上去,可溶表达纯化目的蛋白,并对蛋白的物化性质进行鉴定。选取当前研究应用比较广泛的两个融合标签：SUMO和ELP,对其在促蛋白可溶,标签去除及目的蛋白纯化等方面进行研究。首先,我们利用SUMO表达系统表达三个蛋白：hAPRIL、gelonin和hHAPO,对目的蛋白表达和可溶性进行分析。SUMO-hHAPO融合蛋白以可溶的形式表达,亲和纯化得到高纯度的融合蛋白,SUMO酶能将融合蛋白酶切完全,亲和纯化得到高纯度的hHAPO;质谱和活力分析结果表明重组hHAPO蛋白分子量与天然蛋白的一样,具有高活力。而SUMO-hAPRIL和SUMO-gelonin以最佳表达条件(ArcticExpress(DE3)表达菌,低温、低浓度IPTG)诱导表达后,有少部分融合蛋白以可溶形式表达；上清通过亲和纯化得到融合蛋白能完全被SUMO酶酶切完全,亲和得到最终目的蛋白；在得到的可溶融合蛋白部分,目的蛋白是折叠正确的,这说明了SUMO能促进蛋白以天然的结构折叠可溶表达。ELP系统是由WOOD教授开发的一种新的无需通过介质纯化而获得高纯度蛋白的表达系统。在我们之前的ELP表达实验研究中发现,ELP融合蛋白均以可溶的形式表达。接下来,我们利用ELP表达系统对hAPRIL和gelonin两个蛋白的可溶性表达进行研究。ELP融合蛋白均以可溶性形式表达,相变纯化得到高纯度的融合蛋白；但融合蛋白自动切割及目的蛋白分离纯化不成功,原因可能是：虽然融合蛋白以可溶的形式表达,但可能只是可溶ELP标签本身的聚集使得融合蛋白以可溶的形式表达；但大部分目的蛋白在ELP蛋白的融合下,并没有折叠正确,形成与天然蛋白一样的空间结构,从而大部分融合蛋白不能自动切割去除标签,获得目的蛋白。根据ELP和SUMO表达系统的特点,将两种标签融合起来,建立一种新的在大肠杆菌中促蛋白可溶性高效表达系统。两种融合标签融合表达可能会成为一直新型融合表达蛋白模式或一种趋势。为了验证ELP-SUMO系统的高效促蛋白可溶能力,将SUMO-hAPRIL和SUMO-gelonin亚克隆到pEI表达载体,融合到ELP蛋白C端,构建ELP-SUMO融合表达载体。ELP-SUMO融合蛋白大多数以可溶形式表达,相变纯化得到高纯度融合蛋白。融合蛋白在自动切割溶液中能自动切割完全,表明目的蛋白折叠完全。ELP-SUMO融合蛋白能被SUMO酶酶切完全,亲和纯化得到目的蛋白。质谱结果表明得到的重组蛋白分子量与天然蛋白一样；活力检测也表明目的蛋白具有活力。同时,我们利用ELP-SUMO融合表达系统可溶表达纯化得到LTVGel蛋白,对LTVSPWY对Gel靶向杀伤肿瘤的研究。MTT结果表明,LTV肽能提高Gel对SKOV3肿瘤细胞的杀伤作用(LTVGel, IC50=80nM; Gel, IC50=400nM),对凋亡途径中两个蛋白检测的结果也表明了LTVGel是通过诱导细胞凋亡来杀死肿瘤细胞。更多还原

【Abstract】 Recombinant proteins are used throughout biological and biomedical science. Escherichia coli is the preferred host for rapid and low-cost production of recombinant proteins and peptides for biochemical analysis, therapeutics or structural studies. E. coli heterologous protein production has challenges including protein misfolding and insolubility. Several approaches, including protein fusions, chaperone co-expression, and promoter alterations, have been used to overcome these problems. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. As such, protein fusions have generally been used to improve the expression and solubility of recombinant proteins. No single fusion tag can increase the expression and solubility of all target proteins. However, some fusion tags have been more successful than others in increasing the solubility of many proteins. SUMO tags are emerging as a viable alternative for increasing both the expression and solubility of otherwise hard-to-express proteins.In the present paper, we are focus on developing a new expression system can increase the solubility of target proteins in E.coli. The new system will be used in soluble expression and purification of target protein. Two tags were choosed for the study of soluble ablity, removal of the tag. Firstly, we tested human SUMO as fusion tags for recombinant proteins. Three candidate proteins, hAPRIL、gelonin and hHAPO were expressed as fusion proteins with His6-SUMO. These constructs were expressed in E. coli and evaluated for expression and solubility. Finally, the SUMO-hHAPO fusion protein expressed in a soluble form, cleavage and purification of the fusion protein using the human SUMO expression system is easily accomplished by using a SUMO protease to cleave the fusion tag followed by purification of the hHAPO to homogeneity by Ni-Sepharose column. Mass spectrometry analysis and Cell proliferation assays showed that the recombinant hHAPO with high activity has the same MW with the natural protein. The soluble expression form of the other two fusion construct of SUMO-hAPRIL and SUMO-gelonin can be partly obtained in these expression conditions:ArcticExpress (DE3) strain, low induxtion temperature and low concentration of IPTG. The fusion protein can be complete digested by SUMO protease, the target protein was obtained by affinity chromatography. These results showed thay the SUMO modification is effective.The ELP expression system is developed by WOOD. An expression system is present for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. In our previous experiment, all the target proteins, expressed in a soluble form when fused to ELP. Next, we evaluated the ELP system for expression and solubility of hAPRIL and gelonin. All the proteins, expressed as soluble fusions, were isolated from the protein debirs by the method called inverse transition cycling (ITC); but the efficiency of self-cleavage is low, and the target can not be separated form the fusion protein. The most widely viewed hypothesis for this result is that the seemingly soluble proteins are actually existing as ’soluble aggregates’, held in solution by interactions with the solubility partner, but not in their true, native, soluble form.The solubility and cleavage of the fusion protein using the ELP-SUMO co-expression system is easily accomplished. Using two fusion partner co-expresses with target protein maybe a new expression method in the future. The two proteins of hAPRIL and gelonin were also used in the analysis of ELP-SUMO co-expression system. The ELP-SUMO fusion protein were expressed soluble and with high purity by ITC. The fusion partner can be self-cleavage well in the cleavage buffer showed that the fusion protein folding well that expressed with ELP tag. Finally, we showed that cleavage and purification of the target protein using the ELP-SUMO expression system is easily accomplished by using a SUMO protease to cleave the fusion tag (ELP-SUMO) followed by purification of the target protein to homogeneity by Ni-Sepharose column. Mass spectrometry analysis and Cell proliferation assays showed that the recombinant hHAPO with high activity has the same MW with the natural protein.The ELP-SUMO expression system was also used to purify the LTVGel protein, a LTVSPWY peptide at the N terminal of gelonin. The MTT analysis showed a significant increase in cytotoxic SKOV3 cell by LTVGel compared with Gel. LTVGel kill SKOV3 cell by the mechanism:induction of apoptosis through caspase activation, and then cause cell death.更多还原