融合蛋白CBD-SPA“Z”基因的构建、表达与活性鉴定

针对金黄色葡萄球菌蛋白A(SPA)层析柱的应用问题,将SPA的Z结构域基因与纤维素结合结构域(CBD)重组,并在Z结构域C端引入1个半胱氨酸(Cys),构建并表达出一种新型免疫亲和材料。利用基因工程技术将质粒pEZZ18中的Z基因插入到含有CBD的质粒pET35b(+)质粒中,构建出原核表达载体pET35b(+)-ZCys,经IPTG诱导表达,获得CBD-Protein Z融合表达蛋白。pET35b(+)-Z-Cys在E.coli BL21中正确表达,所表达的融合蛋白具有与哺乳动物IgG抗体结合的生物学活性和与纤维素结合的活性。结果证明CBD-Protein Z蛋白具有Z结构域与CBD结构域的活性,预计能在免疫亲和层析中用于IgG抗体的纯化。     

金黄色葡萄球菌蛋白A串联Z结构域的表达、纯化及其亲和填料制备

目的构建金黄色葡萄球菌蛋白A(Staphylococcal Protein A,简称SPA或蛋白A),串联Z结构域的重组表达克隆,并实现在大肠杆菌中诱导表达,随后纯化获得重组蛋白,制备亲和填料.方法对目的基因进行元和表达的密码子优化,利用PCR扩增方法获得SPA的3个串联Z结构域,并在蛋白的N端加入组氨酸标签.将优化后的基因序列克隆到p ET43.1a(+)表达载体上,获得p ET43.1a(+)-SPA-3Z重组质粒;测序正确的重组质粒转化大肠杆菌感受态细胞BL21(DE3),IPTG诱导表达,通过亲和层析方法纯化表达的重组蛋白,并使用SDS-PAGE鉴定表达蛋白.将纯化获得的SPA纯品偶联到经环氧氯丙烷活化的Sepharose 6B Fast Flow凝胶上.使用兔多抗和腹水单抗对填料进行验证.结果成功构建p ET43.1a(+)-SPA-3Z克隆,纯化的SPA蛋白纯度高.成功将重组SPA偶联到经环氧氯丙烷活化的Sepharose 6B Fast Flow凝胶上.制备的填料在0.1mol/L的Na OH溶液里浸泡30小时,填料亲和性能未见明显变化,这为填料的长期使用提供了可能.将该填料用于抗体纯化,湿胶的多抗结合量可达22.7mg/ml,纯化的腹水单抗纯度在98%以上.结论本研究为SPA的大规模制备提供了基础.     

抗溶葡萄球菌酶N端合成多肽抗体的制备与鉴定

为了制备抗溶葡萄球菌酶N端合成多肽抗体,合成了溶葡萄球菌酶(lysostaphin)分子的3-13位的11个氨基酸的多肽(THEHSAQWLN),并利用戊二醛双功能试剂将人工合成多肤成功地与KLH进行偶联.免疫新西兰兔制备抗lysostaphin合成多肽的抗体,并经亲和层析进行了纯化.对此抗体进行鉴定的结果表明,抗溶葡萄球菌酶合成多肽抗体可与重组溶葡萄球菌酶分子发生特异性反应,并可用于蛋白免疫印迹.该抗体的制备为使用亲和层析纯化溶葡萄球菌酶提供了有用的配基.     

人IgG四亚类对噬菌体展示Ig结合蛋白单结构域随机组合文库的体外进化筛选

金黄色葡萄球菌蛋白A(Staphylococcal protein A,SpA)和链球菌蛋白G(Streptococcal protein G,SpG)是细菌产生的特异结合宿主抗体的细菌免疫球蛋白结合蛋白(Immunoglobulin(Ig)-binding proteins,IBPs)的代表分子。SpA和SpG均包含由多个序列高度同源的结合结构域重复组成的抗体结合区,各单结构域都具有完全的结合IgG的功能。为研究这些单结构域随机组合能否产生具有新结合特性的组合分子,将SpA的A、B、C、D、E以及SpG的B2、B3共7个单结合结构域随机组合构建成噬菌体展示文库后,应用人IgG1、2、3、4为诱饵分子对该文库进行4轮筛选,获得了SpA天然分子中不存在的单结构域排列组合分子D-C。在筛选过程中,阴性对照噬菌体的逐渐减少、展示两个结构域以上的噬菌体比例不断增多,尤其是D-C组合的选择性富集和其随机连接肽的严格筛选都显示了筛选的有效性和D-C组合的重要性。噬菌体ELISA进一步证实D-C与人IgG四亚类的结合能力远强于天然SpA分子。该研究应用分子进化技术首次获得了一种与人IgG四亚类具有结合优势的新型组合分子D-C,不仅可为IgG纯化、制备、检测等方面的应用提供新的候选分子,还为细菌IBP结构功能的进一步研究提供新的手段。     

一种新型免疫亲和层析介质的研究

以国产琼脂糖介质为原料,合成了含肼琼脂糖介质。抗体Fc段的糖基经氧化后,可与此种含肼琼脂糖介质偶联,制成高亲和率的免疫亲和层析介质。与溴化氰法相比,这种对抗体上的糖特异的偶联方法制成的免疫亲和层析介质,容量高、稳定性强。用这种含肼琼脂糖介质分别与肿瘤坏死因子和γ-干扰素等的抗体偶联,制成的免疫亲和层析介质可用于分离纯化相应的基因工程产物抗原。     

葡萄球菌蛋白A活性机制与现代应用

葡萄球菌蛋白A(staphylococcus protein A,SPA)是金黄色葡萄球菌(Staphylococcus aureus)细胞壁上的一种黏连蛋白.经过几十年的研究,由于其对免疫球蛋白IgG的亲和特性,SPA已作为一种工具在抗体的检测、纯化以及疾病诊断中得到了广泛应用.SPA衍生物的构建适应了现代生产的需要,改造后的SPA可以与多种报告分子相偶联,使得免疫诊断更加快速准确.本文综述了SPA基于其免疫学特性,在抗体纯化和现代免疫分析应用中的发展状况及前景.     

抗甲肝病毒基因工程单抗分离纯化与鉴定

为克服血源免疫球蛋白制品的不足,开发了抗甲肝病毒基因工程单克隆抗体anti-HAV IgG。用无血清培养基培养rCHO工程细胞株,上清液经过rProtein A SFF亲和层析→脱盐→离子交换层析→超滤换液纯化后,所得anti-HAV IgG纯度达99%以上,比活性约100IU/mg,anti-HAV IgG活性回收率40%。所纯化的anti-HAV IgG分子量150kD,等电点8.4～9.3。免疫印迹实验证实anti-HAV IgG为人源全抗体分子。亲和层析介质rProtein A SFF确实存在亲和配基脱落问题,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti-HAV IgG各项纯度检测指标均达到我国对基因工程产品的质量要求。     

PTD融和蛋白的原核表达及其抗体制备

目的:为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原核表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础.方法:应用重叠延伸的基因融合技术将LHRH(黄体生成激素释放激素)基因、TAT(HIV-1反式转录激活因子)基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28a-LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni-NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体.结果:表达产物经聚丙烯酰胺凝胶电泳检测,LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12.6%.LTA蛋白经Ni-NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清.结果表明抗体的效价为1: 12800.结论:应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体.     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

密理博ProSep Ultra Plus

ProSep Ultra Plus是一种蛋白A亲和层析介质,是当前市场上动态结合载量及通量最高的一种亲和树脂,是为适应现今低成本、大规模制备高浓度治疗性抗体需求而设计的。     

High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli

Summary An approach to produce ¹³C-and ¹⁵N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40–200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with ¹³C₆-glucose and (¹⁵NH₄)₂SO₄ as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing ¹³C₆-glucose usage. Incorporation levels of ¹³C and/or ¹⁵N isotopes in purified IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20–40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.Abbreviations BPTI bovine pancreatic trypsin inhibitor - DTT dithiothreitol - Gdn-HCl guanidinium hydrochloride - IAA -indole acrylic acid - IGF-II insulin-like growth factor II - PBS phosphate-buffered saline - PDMS plasma desorption mass spectrometry - PFPA pentafluoro propionic acid - RP-HPLC reversed-phase high performance liquid chromatography - Z IgG-binding protein domain derived from staphylococcal protein A.     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.     

The starch-binding domain as a tool for recombinant protein purification

Recombinant protein purification with affinity tags is a widely employed technique. One of the most common tags used for protein purification is the histidine tag (His_tag). In this work, we use a tandem starch-binding domain (SBD_tag) as a tag for protein purification. Four proteins from different sources were fused to the SBD_tag, and the resulting fusion proteins were purified by affinity chromatography using the His_tag or the SBD_tag. The results showed that the SBD_tag is superior to the His_tag for protein purification. The efficient adsorption of the fusion proteins to raw corn starch was also demonstrated, and two fusions were selected to test purification directly using raw starch from rice, corn, potato, and barley. The two fusion proteins were successfully recovered from crude bacterial extract using raw starch, thus demonstrating that the SBD_tag can be used as an efficient affinity tag for recombinant protein purification on an inexpensive matrix.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Abstract Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG₁, IgG₃ and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.     

Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex

Staphylococcal protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). Based on the affinity motif of SpA, we have herein developed a biomimetic design strategy for affinity peptide ligands of IgG. First, according to the distribution of the six hot spots of the SpA affinity motif determined previously, the number of residues that should be inserted into between the hot spots was determined. Cysteine was introduced as one of the middle inserted residues of the peptide for later immobilization. Then, amino acid location was performed to identify other amino acid residues for insertion, leading to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Thereafter, molecular dynamics simulations were performed to validate the dynamics of the affinity interactions between the candidates and IgG, and 14 of them were found to keep high affinities. Finally, the affinity and specificity of the top one ligand FYWHCLDE were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

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.