肌球蛋白轻链激酶CaM结合位点突变体的构建

目的:平滑肌肌球蛋白轻链激酶(myosin light chainkinase,MLCK)具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,本实验利用分子生物学技术构建了肌球蛋白轻链激酶CaM结合位点突变体,并纯化出重组的MLCK表达的蛋白质,为深入研究MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制提供了实验基础.方法:利用野生型MLCK全长的cDNA序列设计CaM结合位点的突变引物,利用PCR技术进行定点突变,获得CaM结合位点的突变体(△CaM/MLCK).在大肠杆茵中表达重组CaM结合位点的突变体(△CaM/MLCK),通过亲和层析及凝胶过滤进行分离纯化重组蛋白,SDS-PAGE检测表达及纯化的重组蛋白.结果:构建重组MLCK钙调蛋白结合位点突变体(△CaM/MLCK),△CaM/MLCK在大肠杆菌中以可溶形式大量表达并得到纯化.结论:成功构建重组MLCK钙调蛋白结合位点突变体(△CaM/MLCK)并获得纯化的表达蛋白质.     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。     

脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

用PCR法克隆出nNOS的CaM结合区基因(nNOS 2455～2988bp),并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为90％以上的重组蛋白．分子量为22kDa,CaM Oveday assay证实该蛋白具有CaM的结合活性。由于所表达的重组蛋白既具有序列特异性又具有CaM的结合活性．因此。可将它作为筛选nNOS特异性抑制肽的靶蛋白,亦可用于特异性抗体的制备。     

多聚精氨酸融合增强型绿色荧光蛋白制备方法及穿膜效果

为了方便细胞穿膜肽R9融合蛋白的可溶性表达及功能上的研究,构建了pSUMO (小分子泛素样修饰蛋白) -R9-EGFP (增强型绿色荧光蛋白) 原核表达载体。分别纯化EGFP及R9-EGFP蛋白后,作用于HepG2,细胞经流式细胞仪及激光共聚焦检测R9细胞穿膜肽的作用效果。实验结果显示在SUMO分子伴侣的作用下,R9-EGFP融合蛋白获得可溶性表达。经流式细胞仪检测,R9细胞穿膜肽可以快速有效的携带目的蛋白进入细胞内部且呈时间、剂量依赖性,大约1.5 h以后荧光强度进入平台期。共聚焦显微镜检测结果表明R9细胞穿膜肽可以有效携带EGFP进入HepG2细胞,并显示主要聚集在细胞浆内。同时体外经肝素抑制实验显示,肝素抑制R9-EGFP穿膜的效率达到50％。这些结果表明,可以利用pSUMO-R9/Ni-NTA表达纯化系统,快速、有效地表达出可溶性多聚精氨酸融合蛋白,同时R9细胞穿膜肽可以有效地携带目的蛋白进入细胞内,为进一步研究多聚精氨酸的穿膜机制提供了基础。     

一种用于肿瘤药物治疗的新型人源性穿膜肽的优化及其应用

细胞穿膜肽(cell-penetrating peptide,CPP)作为一种体内大分子药物跨膜运输载体被广泛研究和应用。中期因子Midkine(MK)是人体的一种带有肝素结合域(heparin-binding domain,HBD)的生长因子。报道了MK中HBD的部分富含碱性氨基酸的基因序列(命名为MK-S0)与绿色荧光蛋白(EGFP)基因融合表达后,能将EGFP有效地转运入胞内,且其穿膜转运效率高于经典穿膜肽Tat。将MK-S0序列进一步突变优化改造得到的midkine-mutantΔ4(MK-Δ4),其穿膜效率比天然序列来源的MK-S0提高16倍以上,且MK-Δ4的穿膜转运作用适用于多种肿瘤细胞。穿膜机制分析研究结果显示,MK-Δ4可与细胞表面硫酸乙酰肝素结合,随之以巨胞饮形式内吞入胞。采用MTT方法检测的细胞生长抑制试验结果显示,连接有MK-Δ4的苦瓜来源的核糖体失活蛋白MAP30比单独的MAP30对HeLa肿瘤细胞的药效可提升5.8倍,大大提高了这种药物蛋白抑杀肿瘤细胞的效果。由此表明,源于MK的这种经过突变改造的MK-Δ4,可作为一种新型高效的细胞穿膜肽,将药物蛋白有效运输到细胞内发挥抗肿瘤效应。     

膜转运蛋白的功能性超表达和纯化

膜转运蛋白结构和功能的研究是功能膜蛋白质组研究中的一个重要内容,而大量蛋白质的分离纯化是进行蛋白质的结构和功能研究的基础.目前,结构和功能膜蛋白质组学相关研究的瓶颈,在于不能有效地超量表达和纯化具有生物活性的膜转运蛋白.影响膜转运蛋白超量表达和纯化的关键因素,包括目标蛋白的拓扑学结构分析和去垢剂的选择.进行膜转运蛋白拓扑学结构的分析,对于构建用于活体表达的重组膜转运蛋白具有指导意义.去垢剂能够稳定去膜状态的膜蛋白,在膜转运蛋白的离体表达和亲和纯化以及包涵体的处理过程中具有重要的作用.本文就目前功能膜蛋白质组学研究中所涉及的有关膜转运蛋白功能性超表达和分离纯化策略及关键技术作一简述.     

亲和标签在重组蛋白表达与纯化中的应用

亲和标签融合技术为重组蛋白的纯化提供了一种简单方便的纯化工具,具有结合特异性高、洗脱条件温和、通用性强、纯化倍数高等显著优点。概述了亲和标签对融合蛋白表达的影响,可以提高重组蛋白的产量,增强重组蛋白的可溶性,促进重组蛋白的正确折叠;回顾了在重组蛋白表达与纯化中广泛使用的几种亲和标签,以及近年来相继出现的几种比较新颖的纯化标签;介绍了亲和标签的组合使用策略,His6-MBP组合标签集合了两个标签的优点,串联亲和纯化可以纯化获得生理条件下的蛋白质复合体;展望了亲和标签未来的发展趋势,认为仍需继续开发性能更加优越、纯化效果更加显著的纯化标签系统。     

灵芝钙调蛋白基因CaM的克隆与功能分析

灵芝是我国传统的食药用真菌,具有广泛的免疫调节功能并含有多种生物活性成分。灵芝酸是灵芝的主要次生代谢产物之一,具有较高商业价值。钙调蛋白（calmodulin,CaM）作为真核生物中重要的Ca ²⁺信使,能够参与生长发育、次生代谢等重要生理过程。本研究通过序列分析发现,灵芝CaM基因序列编码149个氨基酸,与其他物种CaM蛋白高度保守。该蛋白含有4个完整的EF-hand结构域,并且在每个EF-hand结构域中,都含有一个保守的D-x-D基序。进一步构建筛选了灵芝CaM沉默转化子,检测CaM在灵芝生长发育及次生代谢过程中的功能。结果显示,CaM沉默转化子中灵芝酸含量比WT降低约34%。沉默CaM后菌丝生长速率与WT相比降低40%。该结果说明CaM在灵芝生长及次生代谢过程中具有重要作用,为在真菌中研究CaM功能及调控途径提供参考。     

大鼠谷氨酰胺转运蛋白SNAT2-EGFP融合蛋白的表达与鉴定

谷氨酰胺转运蛋白是中枢神经系统中一种重要的中性氨基酸转运蛋白,对谷氨酰胺的跨膜转运十分重要。为了更方便地研究大鼠谷氨酰胺转运蛋白2(SNAT2)在细胞膜上的表达与定位,利用亚克隆技术将增强型绿色荧光蛋白(EGFP)构建于SNAT2的C端,通过菌液PCR、酶切和DNA测序鉴定重组真核表达质粒;将测序正确的重组质粒瞬时转染人胚胎肾细胞(HEK293T cells),用Western blot和激光共聚焦电子显微镜荧光检测技术鉴定SNAT2-EGFP的表达与亚细胞定位。结果表明,SNAT2-EGFP融合蛋白重组质粒在细胞中表达并正确定位于细胞膜上。SNAT2-EGFP融合蛋白重组质粒的成功构建为今后深入研究SNAT2的结构和功能提供了一个有效的工具。     

Tb~(3+)作为荧光探针研究钙调蛋白与拮抗药物的相互作用

本文报导以Tb~(3+)作为荧光探针,研究钙调蛋白(CaM)与其拮抗药物分子间相互作用的机制.所用方法简便、快速、灵敏.CaM的内源荧光研究表明,Tb~(3+)类似于Ca~(2+),也能诱导CaM分子构象发生改化,由于CaM分子中Ca~(2+)的第Ⅲ、Ⅳ结合位点上各有一个Tyr线基,如(?)280nm激发,则发生从Tyr向Tb~(3+)的能量转移,从而导致Tb~(3+)在490和545nm处的特征荧光发射大大加强.本文检测了药物分子与Tb~(3+)-CaM结合对该荧光发射的影响.实验表明,TFP与CaM的高亲和位点处于CaM分子C-末端部位,即含第Ⅲ、Ⅳ结构域的半分子上:丙拮抗药物酸枣仁皂甙A则优先结合在含第Ⅰ、Ⅱ的结构域的另一半分子(?).     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.     

Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase

A Ca2+ -dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+ -ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 amino acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4- to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general.     

A new expression vector for high level protein production,one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7lacO promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15–20 mg/l culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [γ-³²P]ATP and used to probe protein–protein or protein–nucleic acid interactions.     

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flag™, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH₂-terminal seven amino acid residues of human TGF. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag–anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.     

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.     

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.     

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.     

Expression of a Brassic napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein

Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.     

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.