包涵体复性研究进展(英文)

用基因工程技术在大肠杆菌高水平表达重组蛋白时,通常形成无生物活性的包涵体。包涵体在体外经分离、溶解与重折叠后可实现复性,表现为具有生物活性的蛋白。总结了包涵体的相关复性技术,重点介绍重折叠的最新进展情况 。     

鲍曼不动杆菌外膜蛋白34的表达纯化及其生物活性分析

通过基因克隆方法制备鲍曼不动杆菌ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白34(Outer membrance protein 34,Omp34),并分析其生物结构及对HEK293细胞的凋亡作用。采用PCR方法扩增鲍曼不动杆菌ATCC19606Omp34基因,并将其克隆到表达载体p ET28a(+)以构建重组质粒pET28a(+)-Omp34,转化大肠杆菌BL21,IPTG诱导蛋白表达并用镍柱亲和层析纯化蛋白,使用L-精氨酸和还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)可使Omp34蛋白正确复性,生物信息预测Omp34生物结构,利用圆二色谱(Circular dichroism CD)技术测定Omp34的二级结构,通过流式细胞术和Western blot检测Omp34对HEK293细胞的凋亡作用。成功构建表达载体p ET28a(+)-Omp34,并在原核表达体系中高效表达,通过复性得到可溶蛋白。生物信息预测Omp34为β桶状蛋白,圆二色谱证实Omp34结构为β折叠结构,其中α-螺旋含量为12%、β-折叠为39%、β-转角为22%、无规则卷曲为27%。复性的Omp34可使HEK293细胞发生凋亡。通过分子克隆技术成功获得了鲍曼不动杆菌外膜蛋白34,并解析出其二级结构,证实其可以导致HEK293细胞凋亡,这为将来进一步研究鲍曼不动杆菌外膜蛋白34的生物学功能及其耐药机制奠定了基础。     

重组蛋白包涵体的复性研究

重组蛋白在大肠杆菌中的高表达往往形成不可溶、无生物活性的包涵体,需经过变性溶解后,在适当条件下复性形成天然的构象,才可恢复其生物活性．变复性实验是建立在对蛋白质体外折叠机制的了解的基础上．根据近年来对蛋白质折叠机制的认识和重组蛋白包涵体在复性方面的主要进展,论述以下3个方面的内容：1)蛋白质在细胞内的折叠机制;2)蛋白质体外折叠机制;3)蛋白质复性的策略和方法．     

弧菌外膜蛋白OmpU的免疫交叉反应性和交叉保护性北大核心CSCD

【目的】通过对弧菌外膜蛋白Omp U的克隆、表达以及免疫学特性分析,明确外膜蛋白Omp U是否为弧菌的共同抗原,并具有免疫交叉反应性和交叉保护性。【方法】对弧菌外膜蛋白omp U基因进行克隆和生物信息学分析。分别制备副溶血弧菌、溶藻弧菌、创伤弧菌、拟态弧菌和霍乱弧菌的Omp U重组蛋白抗血清,对Omp U的免疫交叉反应特性以及抗原表位定位情况进行比较分析。以霍乱弧菌的Omp U重组蛋白免疫小鼠后,再以多种弧菌进行攻毒,分析其交叉免疫保护作用。【结果】外膜蛋白Omp U在弧菌种内和种间相似性分别为73.0%–100%和58.6%–89.0%,并至少存在9个保守的B细胞抗原表位。Omp U重组蛋白抗血清在弧菌种内和种间均产生显著的免疫交叉反应,识别弧菌中分子量35–40 k Da的同源蛋白。副溶血弧菌ATCC17802、创伤弧菌ATCC27562和拟态弧菌ATCC33653来源的Omp U重组蛋白抗体能识别供试菌株,提示这些菌株的Omp U抗原表位定位于细胞表面。Omp U重组蛋白对免疫后的小鼠具有交叉免疫保护作用,攻毒实验后小鼠相对存活率(RPS)为43.0%–100%。【结论】上述结果表明,外膜蛋白Omp U是弧菌中一种保守的共同抗原,具有免疫交叉保护性,可以作为弧菌广谱疫苗的候选抗原。     

菌丝霉素MP1106融合蛋白的复性及纯化方法

旨在建立高效、快捷的菌丝霉素衍生物MP1106大肠杆菌表达系统。通过基因融合的方式构建生物表面活性剂-菌丝霉素衍生物(DAMP_4-MP1106)融合蛋白表达载体,在大肠杆菌中进行表达;并对目的蛋白MP1106进行分离纯化和分子内二硫键鉴定。结果显示,融合蛋白DAMP_4-MP1106在大肠杆菌中以包涵体的形式成功表达,表达产物在变性条件下经Ni~(2+)-NTA亲和层析纯化;经检测分析,摇瓶中DAMP_4-MP1106的发酵产量为118 mg/L,纯度为94.7%;采用96孔板筛选并建立复性方法,获得水溶性融合蛋白DAMP_4-MP1106;并经TEV蛋白酶酶切以及二次Ni~(2+)-NTA亲和层析纯化,可获得纯度为99%的抗菌肽MP1106 18mg/L,回收率达到了38.4%。通过简捷方法快速鉴定分子内的二硫键,初步证实了抗菌肽MP1106完成了分子内结构正确折叠。建立了高效快捷的菌丝霉素大肠杆菌表达系统。     

大肠杆菌外膜蛋白酶T及其突变体的表达、复性及生物活性分析

外膜蛋白酶T(Outer-membrane protease T,OmpT)是定位于大肠杆菌外膜,具有高度底物特异性的蛋白水解酶。本文旨在建立克隆表达膜蛋白OmpT和体外复性的方法,考察其蛋白酶活性。首先以大肠杆菌基因组DNA为模板,PCR扩增ompT基因,连接至pET28a(pET-ompT),引入点突变Asp85Ala,构建表达质粒pET-ompT85。然后将两种重组质粒转化入BL21(DE3),均以包涵体形式大量表达。纯化后的蛋白经稀释法复性,并加入粗制脂多糖(Lipopolysaccharide,LPS)恢复蛋白酶活性。通过SDS-PAGE、鱼精蛋白水解试验及生长曲线观察表明,重组蛋白OmpT在体外能水解抗菌肽鱼精蛋白和兔肌肉肌酸激酶,而OmpT突变体则无上述功能。上述结果表明本文获得了具有蛋白水解酶功能的重组蛋白OmpT,该蛋白在体外可保护大肠杆菌抵抗鱼精蛋白的杀菌作用。     

一种从大肠杆菌包涵体中分离纯化GST-TRAF6融合蛋白的方法

利用8 mol/L尿素溶液对表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白进行变性,通过逐级稀释复性的方法对尿素溶解后的GST-TRAF6融合蛋白进行复性,将复性后的GST-TRAF6融合蛋白进一步利用谷胱甘肽琼脂糖树脂亲和层析的方法进行分离纯化,将分离纯化后的蛋白通过Western blot方法进行验证,最后利用体外泛素化反应检测经包涵体变性、复性和纯化后的GST-TRAF6融合蛋白的生物学活性。经过包涵体变性、梯度稀释复性和谷胱甘肽琼脂糖树脂亲和层析3个步骤后纯化得到纯度达90%以上、浓度为396 ng/μL的蛋白质溶液。利用GST蛋白作为对照,经Western blot验证表明,纯化得到的蛋白确为GSTTRAF6融合蛋白。进一步利用体外泛素化反应分析其泛素连接酶活性发现,17 ng/μL浓度的GST-TRAF6融合蛋白能够以泛素分子作为底物在5 min内快速催化自由泛素链的生成。结果表明,表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白经尿素变性溶解后能够成功复性并分离纯化,在溶解性改变的同时恢复了其泛素连接酶活性。为从大肠杆菌包涵体中大规模分离纯化蛋白质提供了一种新的复性方法。     

重组融合蛋白Trx-IFN-CSP复性工艺研究

旨在建立并优化融合蛋白Trx-IFN-CSP的复性工艺。对重组融合Trx-IFN-CSP进行体外复性研究,考查p H、温度、蛋白浓度、氧化还原体系及辅助复性小分子等复性条件对融合蛋白重折叠的影响。结果显示,适合Trx-IFN-CSP复性的方法为,反复冻融联合超声破菌获得包涵体;用含有1%Triton X-100、2 mol/L尿素、2%DOC洗涤液初步纯化包涵体;再用6 mol/L盐酸胍溶解液变性包涵体;脉冲加样稀释变性液后4℃条件下梯度透析复性,使用L-Arg辅助复性。经肠激酶切去Trx标签后,每升发酵液最终获得110-130 mg肝靶向干扰素,每批蛋白纯度都在95%以上,比活性在1.9-2.4×108 U/mg之间,制备工艺稳定。     

蛇毒金属蛋白酶Alfimeprase在大肠杆菌中的表达、纯化及活性检测

目的:应用大肠杆菌表达系统表达纤溶性蛇毒金属蛋白酶Alfimeprase。方法:运用PCR技术扩增人工合成的目的基因,引入Nde Ⅰ、EcoR Ⅰ双酶切位点;目的基因经Nde Ⅰ、EcoR Ⅰ双酶切后,克隆到pET22b载体上,然后将重组质粒转化大肠杆菌BL21(DE3)进行胞内表达,对获得的包涵体进行体外透析复性;应用纤维蛋白原水解法、纤维蛋白平板法和纤维蛋白层叠法检测复性后蛋白的降纤活性。结果:目的蛋白以包涵体形式在大肠杆菌中获得高表达,复性后的蛋白具有降解纤维蛋白(原)的活性。结论:Alfimeprase包涵体可以通过复性获得活性产物。     

革兰氏阴性细菌β-桶状结构外膜蛋白体外折叠的研究进展

除了细胞质膜,革兰氏阴性细菌细胞还有一层组成细胞壁的外膜(Outer Membrane)。膜蛋白是外膜的主要组成成分之一,绝大多数外膜蛋白是由反向平行的β-折叠(β-Strands)通过相邻的氢键结合形成的β-桶状结构蛋白(β-Barrel Proteins)。这些蛋白既可作为通道蛋白、转运蛋白、酶、受体、毒力因子,也可作为结构蛋白发挥稳定外膜的重要作用,它们是否正确折叠并整合到外膜对革兰氏阴性细菌的生存至关重要。大多数外膜蛋白易于重组表达和体外重折叠(in vitro refolding),并且折叠状态可通过多种方法测定,因此β-桶状结构外膜蛋白被当着模式蛋白来研究各类生物和非生物因子对膜蛋白折叠的影响,是膜蛋白研究的一大热点。本文将从β-桶状结构外膜蛋白体外折叠的研究方法和影响折叠的因素角度对近年相关研究进展进行综合述评,最后总结了外膜蛋白体外折叠模式,并结合作者的相关研究结果和观点对该领域的研究前景进行了展望。     

Eicosapentaenoic acid facilitates the folding of an outer membrane protein of the psychrotrophic bacterium, Shewanella livingstonensis Ac10

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), are found in various cold-adapted microorganisms. We previously demonstrated that EPA-containing phospholipids (EPA-PLs) synthesized by the psychrotrophic bacterium Shewanella livingstonensis Ac10 support cell division, membrane biogenesis, and the production of membrane proteins at low temperatures. In this article, we demonstrate the effects of EPA-PLs on the folding and conformational transition of Omp74, a major outer membrane cold-inducible protein in this bacterium. Omp74 from an EPA-less mutant migrated differently from that of the parent strain on SDS-polyacrylamide gel, suggesting that EPA-PLs affect the conformation of Omp74 in vivo. To examine the effects of EPA-PLs on Omp74 protein folding, in vitro refolding of recombinant Omp74 was carried out with liposomes composed of 1,2-dipalmitoleoyl-sn-glycero-3-phosphoglycerol and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1:1molar ratio) with or without EPA-PLs as guest lipids. SDS-PAGE analysis of liposome-reconstituted Omp74 revealed more rapid folding in the presence of EPA-PLs. CD spectroscopy of Omp74 folding kinetics at 4°C showed that EPA-PLs accelerated β-sheet formation. These results suggest that EPA-PLs act as chemical chaperones, accelerating membrane insertion and secondary structure formation of Omp74 at low temperatures.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.     

Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans

The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules. After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin. The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification). Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis. Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography. Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA.     

Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism

Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 A from the bilayer center. In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 degrees C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 A from the center after refolding was complete. In contrast Trp-7 approached the 10 A distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four beta-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.     

Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.     

Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis. The outer membrane protein A (OmpA) assembles a beta-barrel structure having four surface-exposed loops in E. coli outer membrane. OmpA of meningitis-causing E. coli K1 is shown to contribute to invasion of the human brain microvascular endothelial cells (HBMEC), the main cellular component of the blood-brain barrier (BBB). However, the direct evidence of OmpA protein interacting with HBMEC is not clear. In this study, we showed that OmpA protein, solubilized from the outer membrane of E. coli, adhered to HBMEC surface. To verify OmpA interaction with the HBMEC, we purified N-terminal membrane-anchoring beta-barrel domain of OmpA and all surface-exposed loops deleted OmpA proteins, and showed that the surface-exposed loops of OmpA were responsible for adherence to HBMEC. These findings indicate that the OmpA is the adhesion molecule with HBMEC and the surface-exposed loops of OmpA are the determinant of this interaction.     

Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of ∼ 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163-169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of (R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.