链霉亲和素纯化和鉴定方法的研究

对链霉亲和素进行纯化、鉴定,采用冷钝化的方法去除培养液中大部分杂蛋白,用亲和层析法从链霉菌L-183的培养液中纯化链霉亲和素（SA）,经试验,SA回收率为75％～85％。鉴定表明,自制SA的分子量为74．5kD,每分子SA可结合3．2个生物素分子,活性为11．2U／mg,pI为7．4。自制SA各项生物学性质与文献报道相符。     

碱性磷酸酶标记链霉亲和素

碱性磷酸酶标记链霉亲和素（AP-SA）是酶放大时间分辨荧光免疫分析（EATRFIA）通用的、最关键的试剂.报告了AP-SA的戊二醛二步标记方法.用自行研制的荧光发展溶液和AP的底物5-氟水杨酸磷酸酯测定了标记物AP-SA的特性,AP的标记回收率为38.7%;AP-SA稀释200～12 800倍时,稀释倍数和Tb的信/噪比呈线性关系;至少在两个月内,AP-SA的酶活性是稳定的.     

抗癌胚抗原嵌合重链与核心链霉亲和素基因的融合及 …

将抗癌胚抗原（ＣＥＡ）单克隆抗体的重链可变区与人的恒定区（Ｃγ３）连接,制备抗癌胚抗原嵌合重链用于放免治疗及其他导向治疗,可减少人抗鼠抗体反应（ＨＡＭＡ）。为纯化及核素标记抗体,将嵌合重链基因与核心链霉亲和素基因融合。融合基因在大肠杆菌得到高效表达,表达量占菌体总蛋白的２４％。ＳＤＳ－ＰＡＧＥ和蛋白质印迹图谱显示表达产物分子量为７０ｋＤ,与其基因编码蛋白质的理论推算值相符。以ＨＲＰ标记的生物素作为     

抗癌胚抗原嵌合重链与核心链霉亲和素基因的融合及表达

将抗癌胚抗原（ＣＥＡ）单克隆抗体的重链可变区与人的恒定区（Ｃγ３） 连接, 制备抗癌胚抗原嵌合重链用于放免治疗及其他导向治疗, 可减少人抗鼠抗体反应（ＨＡＭＡ） 。为纯化及核素标记抗体, 将嵌合重链基因与核心链霉亲和素基因融合。融合基因在大肠杆菌得到高效表达, 表达量占菌体总蛋白的２４ ％ 。ＳＤＳＰＡＧＥ 和蛋白质印迹图谱显示表达产物分子量为７０ ｋＤ, 与其基因编码蛋白质的理论推算值相符。以ＨＲＰ标记的生物素作为抗体进行蛋白质印迹, 在７０ ｋＤ 处可见表达条带, 表明融合蛋白能特异性地与生物素结合, 使嵌合重链经生物素柱进行廉价纯化成为可能。表达产物在胞内主要以不溶性的包含体存在, 经变性和复性处理后, ＲＩＡ 表明表达产物具有结合其特异性抗原ＣＥＡ的能力。     

抗癌胚抗原单链抗体基因与核心链霉亲和素基因融 …

将抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合插入昆虫杆状病毒供体质粒ｐＦａｓｔＢａｃＨＴａ中,在粉纹夜蛾Ｔｎ－５Ｂ１－４细胞中进行表达。ＳＤＳ－ＰＡＧＥ分析结果表明,表达产物分子量为４１ｋＤ左右,Ｗｅｓｔｅｒｎ印迹分析结果表明,以ＨＲＰ标记的生物素进行蛋白质印迹在４１ｋＤ处可见表达条带,表明融合蛋白能特异性的与生物素结合,放射免疫分析表明重组杆状病毒表达产生的ＳｃＦｖ－ＣＳ蛋白能特异性结合癌胚     

生物素—链霉亲和毒免疫学法测定地高辛的研究

采用国产链霉亲和素直接包被塑料板孔,生物素标记抗体,建立的竞争酶联免疫吸附试验的方法测定血中地高辛浓度,其测定灵敏度为０．９６４μｇ／Ｌ最低检测限为０．２２５１μｇ／Ｌ,测定三份低,中、高浓度的血甭标本,批内变异系数为８．９％、５．９％、２．４％;批间变民系数为１５．８％、１０．１％、９．２％,测定回收率在８９．１－１０７．２２％之间,此法与ＦＰＩＡ方法相关良好。     

链霉亲和素纯化和鉴定方法的研究*

对链霉亲和素进行纯化、鉴定,采用冷钝化的方法去除培养液中大部分杂蛋白,用亲和层析法从链霉菌L-183的培养液中纯化链霉亲和素(SA),经试验,SA回收率为75%~85%。鉴定表明,自制SA的分子量为74.5kD,每分子SA可结合3.2个生物素分子,活性为11.2U/mg,pI为7.4。自制SA各项生物学性质与文献报道相符。     

链霉菌ZG0429的分类鉴定与链霉亲和素的分离纯化研究

从土壤中筛选得到1株高产链霉亲和素的放线菌ZG0429,根据形态观察、培养特征、生理生化鉴定以及16S rRNA序列分析,初步判定该菌株为链霉菌属中的淡紫灰链霉菌(Streptomyces lavendulae)。经发酵,ZG0429的链霉亲和素产量可达201.0mg/L。进一步采用硫铵沉淀和凝胶过滤层析纯化,链霉亲和素的回收率为76.87%,纯度可以达到97.03%。该方法简单易行,成本低廉,可得到高产量、高纯度、高活性的目的蛋白,为链霉亲和素发酵产品的大规模纯化提供了依据。     

生物素-链霉亲和素免疫学法测定地高辛的研究

采用国产链霉亲和素直接包被塑料板孔,生物素标记抗体,建立的竞争酶联免疫吸附试验的方法测定血中地高辛浓度.其测定灵敏度为0.0964 μg/L,最低检测限为0.2251 μg/L,测定三份低、中、高浓度的血清标本,批内变异系数为8.9%,5.9%,2.4%;批间变异系数为15.8%,10.1%,9.2%,测定回收率在89.1%～107.22%之间,此法与FPIA方法相关良好(r=0.9488).     

抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合在昆虫细胞中的表达

将抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合插入昆虫杆状病毒供体质粒 pFastBacHTa中 ,在粉纹夜蛾Tn 5B1 4细胞中进行表达。SDS PAGE分析结果表明 ,表达产物分子量为 4 1kD左右 ,Western印迹分析结果表明 ,以HRP标记的生物素进行蛋白质印迹在 4 1kD处可见表达条带 ,表明融合蛋白能特异性的与生物素结合 ,放射免疫分析表明重组杆状病毒表达产生的ScFv CS蛋白能特异性结合癌胚抗原     

The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide – instead of D-biotin recognized by the natural protein – to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.     

串联亲和纯化标签分类与优化

质谱技术高速发展,检测灵敏度不断提高,但区分特异性与非特异性相互作用仍然是研究相互作用蛋白的瓶颈,获得高纯度的蛋白复合体是鉴定相互作用蛋白的限制性因素。近年来串联亲和纯化（TAP）技术的产生和发展有效解决了相互作用蛋白鉴定中的特异性问题。TAP技术是将N端或C端TAP标签与目的蛋白融合并导入靶细胞进行表达,裂解细胞释放融合蛋白,在接近生理状态下利用标签两步特异性亲和洗脱得到蛋白复合体。其中,TAP标签蛋白的选择和优化是该技术成功的关键。     

Signal Peptide does not Inhibit Binding of Biotin to Streptavidin

Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.     

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: application to Bruton's tyrosine kinase

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG‐binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin‐binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three‐tag system comprised of CBP, streptavidin‐binding peptide (SBP) and hexa‐histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP‐His₆ purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems.     

串联亲和纯化技术及其应用

串联亲和纯化(tandemaffinitypurification,TAP)是一种能快速研究体内蛋白质相互作用的新技术,经过两步特异性亲和纯化,可快速得到生理条件下与靶蛋白质存在真实相互作用的蛋白质。TAP方法最初用于酵母中,因其具通用性、高效性、高纯度及假阳性低等特点得到了快速发展,至今已成功运用于许多其他生物。现主要介绍TAP方法的原理、TAP标签及其在不同物种中的应用。     

一种改进的融合蛋白亲和层析纯化方法

用马铃薯淀粉柱可以直接分离麦芽糖结合蛋白-乳酸脱氢酶辅酶结合结构域融合蛋白,并得到满意的结果.它提纯的程度和吸附量都和商品交联直链淀粉亲和层析柱相比拟,但是成本却要低很多,而且从市场上买来的马铃薯淀粉就可以应用.它可以成为大规模生产的一种工艺路线.     

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N'-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.     

转铁蛋白受体单链抗体与链亲和素融合基因的克隆及其在大肠杆菌中的可溶性表达

目的:转铁蛋白受体特异性富含于血脑屏障和肿瘤细胞的表面,是当前中枢神经系统疾病和肿瘤治疗中定向转运的重要靶标。拟获得在大肠杆菌中能高效可溶表达的转铁蛋白受体单链抗体与链亲和素(SA)的重组融合蛋白。方法:根据GenBank数据库报道的SA的核苷酸序列分段合成基因,连接后经PCR获得完整的基因片段,插入pGEM-T载体中测序。将序列正确的SA基因与大鼠转铁蛋白受体单链抗体基因ox26-scFv分别插入原核表达载体pTIG-Trx中,构建重组表达克隆pTIG-Trx/scFv-SA,并在大肠杆菌中诱导表达。ELISA检测融合蛋白的生物学活性。结果:对pGEM-T/SA克隆的测序结果显示,合成的SA基因与文献报道相符。重组融合蛋白在大肠杆菌中获得了可溶性表达,约占菌体上清总蛋白量的30%;ELISA结果表明该融合蛋白具备与转铁蛋白受体和生物素的结合的双重活性。结论:有活性的重组融合蛋白的获得为构建一个通用性的以转铁蛋白受体介导的血脑屏障和肿瘤转运靶向载体打下了基础。     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

串联亲和纯化（TAP）技术在蛋白质组学中的应用

蛋白质是各种生命活动的主要执行者,因此构建蛋白质相互作用的网络图对于准确理解蛋白质功能、揭开各种细胞活动的奥秘十分重要.串联亲和纯化（TAP）,是近年来发展出来的一种能够快速研究在生理条件下蛋白质相互作用,揭示蛋白质复合体相互作用网络的新技术,已成为研究蛋白质组学的一个重要工具.随着该技术的不断完善,TAP技术在认识蛋白质相互作用的过程中必将发挥越来越重要的作用.