人酪酪肽及其衍生物的克隆、表达及活性测定

酪酪肽(peptide tyrosine tyrosine, PYY)是存在于机体肠道的肽类激素,有 PYY_1-36和PYY_3-36两种形式,后者可以降低个体食欲并减少食物摄入。分别通过人工合成基因和PCR定点突变的方法,得到PYY_3-36衍生物PYY_3-36-Gly³⁷及PYY_3-36的基因,然后克隆到pET32a(+)表达载体中,转化大肠杆菌并进行诱导表达。通过亲和层析得到融合蛋白,经肠激酶酶切和二次亲和层析得到目的多肽。在昆明鼠体内检测二者生物活性,结果显示剂量为800μg/kg时PYY_3-36及PYY_3-36-Gly³⁷均可抑制昆明鼠的摄食,且抑制作用可达9h,而PYY_3-36-Gly³⁷组的平均抑制率可达50%,明显高于PYY_3-36。     

利用原核表达系统一步法制备生物素标记蛋白质

传统的蛋白质生物素标记多采用体外化学修饰法,涉及生物素和蛋白质的活化、透析和纯化等多种处理,该方法步骤繁琐,且对目的蛋白的损失较大。本实验利用原核共表达质粒pCDFDuet-1,将含有6个组氨酸标签的人己糖苷酶D(hexosaminidase D,HexD)的cDNA与生物素受体多肽(biotin acceptor peptide,BAP)DNA进行PCR拼接,连入pCDFDuet-1的多克隆位点1(multiple cloning site1,MCS1);将以大肠杆菌Trans5α基因组为模板克隆得到的生物素连接酶(biotin ligase,BirA)基因连入MCS2,构建重组质粒pCDFDuet-hexD-BAP-birA。初步验证后将该质粒转化至大肠杆菌BL21(DE3)pLysS中,利用0.1 mmol/L的IPTG和80μmol/L的生物素进行诱导表达,采用Ni-NTA亲和层析和超滤对HexD进行纯化,SDS-PAGE检测分子量的大小(60 kDa)和纯度(90%以上)。以anti-HexD和链霉亲和素-HRP为抗体,Western blot检测发现,HexD-BAP表达正确,且被生物素标记;同时以4-MU-O-GalNAc为荧光底物,检测到生物素化标记HexD的糖苷酶活性为3.6 nmol/(min·μg),与未标记HexD的活性(3.06 nmol/(min·μg))相当。结果表明,可以利用BirA及其受体多肽,通过共表达质粒pCDFDuet-1,一步转化、表达和纯化,在大肠杆菌中进行外源蛋白的表达和生物素标记,且不改变目的蛋白的生物活性,可应用于免疫标记、互作蛋白的捕获等生物学研究。     

生物素化膜联蛋白V的构建及在细胞凋亡研究中的应用

我们利用基因工程的方法在大肠杆菌中制备了生物素化的膜联蛋白Ｖ。为此，我们构建了膜联蛋白Ｖ与乙酰Ｃ０Ａ羧化产生物素受体肽ＤＣＰ８７的融合基因，并在大肠力中进行了表达。表达产物经ＨＰＬＣ检测，我们发现大约有６０％的融合表达产物在大肠杆菌内进行了生物素化。生物素化的膜联蛋白Ｖ经抗生物素蛋白亲和层析化后用于吗啡诱导的下丘脑神经元细胞的凋亡检测，取得了理想的结果。     

基于亲和素-生物素双夹心体系测定外源性蛋白血清动态浓度的非抗体依赖新方法

建立一种非抗体依赖的检测外源性蛋白多肽分子代谢动力学血清浓度及动态变化的新方法．链亲和素为捕获分子,加入待测生物素标记蛋白或合成肽,辣根过氧化物酶标记链亲和素为检测分子,构成链亲和素-生物素标记大分子-酶链亲和素的双夹心体系,并进行特异性、敏感性、准确性及稳定性评价．本检测体系灵敏度高,可达0.3125 μg/L,且可通过改变亲和素包被浓度调整检出敏感度与检测范围．准确性回收率为97.82%～107.92%,批内、批间变异系数分别< 5.76% 和< 8.42%,并成功应用在生物素标记人血清白蛋白(biotin-HSA)与生物素标记鸡卵黏蛋白(biotin-OVM)的小鼠血清浓度动态检测．本方法不依赖抗体与放射性核素,可望在外源性蛋白多肽大分子及其药物的体内定量检测和代谢动力学研究中广泛应用．     

抗心律失常肽抗体制备及放射免疫分析法

将抗原性很弱的半抗原 AAP 与牛甲状腺球蛋白偶联,采用淋巴结免疫法制得免抗 AAP 抗体,效价为1:13600.AAP 分子结构中没有可与 ¹²⁵I发生反应的酪氨酸或组氨酸残基,我们用3-(4-羟苯基)丙酸-N 琥珀酰胺酯做连接剂,通过酰氨键将酯连在 AAP 肽的末端氨基上,再将 ¹²⁵I 标记在酯的羟苯基的2,5位置上,成功地建立了 AAP 放射免疫分析法.     

生物素化荧光素酶的克隆表达及其固定化研究

为了在体内实现萤火虫荧光素酶的生物素酰化修饰,我们将大肠杆菌中编码生物素羧基载体蛋白（biotin carboxyl carrier protein,BCCP）C端87个氨基酸的功能域基因融合到萤火虫(Pyrocoelia pectoralis)荧光素酶cDNA的末端。经大肠杆菌生物素合成酶（biotin holoenzyme synthetase）的催化,生物素与BCCP上特定的赖氨酸（Lys）残基共价结合,由此与BCCP融合的萤火虫荧光素酶间接实现了生物素化的修饰。利用生物素与配体亲和素或链霉亲和素的特异性耦合,可以将荧光素酶固定到亲和素或链霉亲和素包被的磁珠上,从而使荧光检测的应用更加灵活和方便。本文将就生物素化荧光素酶的克隆、表达以及功能检测进行具体讨论。     

短肽蝎毒素的结构分类与功能特征

大量的资料已证实蝎毒中主要致死成分是一类由60～70个残基组成, 选择性地作用于电压门控Na⁺通道的长肽毒素.另一类由30～40个残基组成的短肽蝎毒素,由于其具有结构致密,易于合成改造的优点,特别是具有选择性地阻遏K⁺或Cl^－通道的特异药理功效,近年来倍受学术界的关注,并在结构与功能方面取得了很大的研究进展.     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

抗生物素蛋白结合蛋白的发现及分离纯化

本文报道了在日本血吸虫感染的兔血清及日本血吸虫卵中,存在一种能与抗生物素蛋白（又称亲和素）专一性结合的蛋白质,称为抗生物素蛋白结合蛋白。该蛋白质与亲和素的结合与生物素及亲和素的糖链部分无关,并能在高ＰＨ、高浓度盐及去污剂等条件下与亲和素结合。利用ＤＥＡ－５２离子交换柱及偶联有亲和素的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱,从日本血吸虫感染的兔血清中,分离纯化了该蛋白质。提纯的亲和素结合蛋白在ＳＤＳ－ＰＡ     

In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.     

CdTe nanocrystal-based electrochemical biosensor for the recognition of neutravidin by anodic stripping voltammetry at electrodeposited bismuth film

CdTe quantum dots (QDs)-based electrochemical sensor for recognition of neutravidin, as a model protein, using anodic stripping voltammetry at electrodeposited bismuth film is presented. This biosensor involves the immobilization of the captured QDs conjugates which was dissolved with 1M HCl solution to release cadmium ions and metal components were quantified by anodic stripping voltammetry after a 3-min accumulation at -1.2V on bismuth-film electrode (BiFE) of the biotin, served as recognition element, onto the gold surface in connection with a cysteamine self-assembled monolayer. The modification procedure was characterized by electrochemical impedance spectroscopy and atomic force microscopy. We exploit QDs as labels for amplifying signal output and monitoring the extent of competition process between CdTe-labeled neutravidin and the target neutravidin for the limited binding sites on biotin. As expected for the competitive mechanism, the recognition event thus yields distinct cadmium stripping voltammetric current peak, whose response decreases upon increasing the level of target neutravidin concentrations. Under optimal conditions, the voltammetric response is highly linear over the range of 0.5-100 ngL(-1) neutravidin and the limit of detection is estimated to be 0.3 ngL(-1) (5 nM). Unlike earlier two-step sandwich bioassays, the present protocol relies on a one-step competitive assay, which is more accurate and sensitive, showing great promise for rapid, simple and cost-effective analysis of protein.     

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.     

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

Biotinylation facilitates the uptake of large peptides by Escherichia coli and other gram-negative bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

A novel TNFalpha antagonizing peptide-Fc fusion protein designed based on CDRs of TNFalpha neutralizing monoclonal antibody

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFalpha neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFalpha was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFalpha coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFalpha and also inhibit the TNFalpha-induced cytotoxicity on L929 cells. The TNFalpha antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFalpha antagonists.     

Biotin transport by a biotin-deficient strain of escherichia coli

Biotin uptake has been investigated using an Escherichia coli biotin requiring auxotroph grown under biotin-deficient conditions. This strain accumulated biotin in the free and bound form. In agreement with a previous report by O. Prakash and M.A. Eisenberg (J. Bacteriol. 120 (1974) 785–791), the biotin entry proved to be an active process which depended on an energy source and was inhibited in the presence of uncouplers. The kinetic parameters have been determined (K_M = 0.05 μM, V_max = 7 pmol/min per mg dry weight). The pool of free biotin could be readily exchanged with external biotin and decreased to a very low level in the absence of an energy source. The use of several biotin analogues revealed that this transport system was quite specific for biotin: slight modifications, for instance in the valeric chain. lowered drastically the affinity for the carrier.     

Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.     

Surface expression of epithelial Na channel protein in rat kidney

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface beta and gammaENaC subunits were assayed by immunoblot. Surface alphaENaC was assessed by membrane fractionation. Most of the gammaENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.