Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

Biosynthesis of Biotin in Microorganisms VI. Further Evidence for Desthiobiotin as a Precursor in Escherichia coli

Biotin auxotrophs were isolated from Escherichia coli K-12. One of the mutants was unable to grow on desthiobiotin and accumulated a large amount of a vitamer in medium when growing on an optimal concentration of biotin. The production of the vitamer was inhibited in the presence of an excess amount of biotin. The vitamer was identified as desthiobiotin on the basis of biological activities, avidin combinability, and chromatographic characteristics. The mutant lacked the ability to convert desthiobiotin to biotin. These results further support the hypothesis that desthiobiotin is a normal intermediate in the biosynthesis of biotin in E. coli.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Lentiviral transgenesis

Transgenic animals are relevant for many fields of modern biomedicine and agriculture. However, the inefficiencies of the presently available techniques – DNA microinjection and retroviral gene transfer – have led to an explosion of costs for transgenics especially in farm animals. The recent success in transferring genes to early embryos of different species (mouse, rat, pig, cattle) by viral vectors derived from lentiviruses, has established lentiviral transgenesis as an exciting alternative to the classical method of DNA microinjection. In addition, lentiviral vectors can be used to transfer genes into embryonic stem cells. Due to its high efficacy and versatility, lentiviral transgenesis should have a big impact on transgenic research.     

Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification

The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.     

Synthesis of Desthiobiotin from 7,8-Diaminopel-argonic Acid in Biotin Auxotrophs of Escherichia coli K-12

The synthesis of desthiobiotin from 7,8-diaminopelargonic acid (DAP) was demonstrated in resting cell suspensions of Escherichia coli K-12 bioA mutants under conditions in which the biotin locus was derepressed. The biosynthetically formed desthiobiotin was identified by chromatography, electrophoresis, and by its ability to support the growth of yeast and those E. coli biotin auxotrophs that are blocked earlier in the biotin pathway. Optimal conditions for desthiobiotin synthesis were determined. Desthiobiotin synthetase activity was repressed 67% when partially derepressed resting cells were incubated in the presence of 3 ng of biotin per ml. Serine, bicarbonate, and glucose stimulated desthiobiotin synthesis apparently by acting as sources of CO(2). The results of this study are consistent with an earlier postulated pathway for biotin biosynthesis in E. coli: pimelic acid --> 7-oxo-8-aminopelargonic acid --> DAP --> desthiobiotin --> biotin.     

慢病毒介导的HTRA1 基因稳定过表达人脑血管平滑肌细胞株的建立

目的:构建并包装针对HTRA1基因以及其1091TC突变基因(HTRA1-Mut)的过表达慢病毒载体,以及建立稳定表达HTRA1及HTRA1-Mut基因的人脑血管平滑肌细胞(HBVSMC)株。方法:采用RT-PCR方法扩增HTRA1及HTRA1-Mut基因片段并将其连接于GV287载体质粒,采用慢病毒包装三质粒系统(GV287/p Helper 1.0/p Helper 2.0)转染293T细胞,收集富含慢病毒颗粒的细胞上清液并标定病毒滴度,慢病毒感染经培养和鉴定的HBVSMC细胞株。结果:成功构建含HTRA1及HTRA1-Mut基因的慢病毒重组载体,PCR鉴定阳性的克隆进行测序和BLAST比对分析显示与源基因序列一致,并能够有效的感染并在293T细胞中表达。表达载体包装后测定病毒滴度为:2E+8 TU/mL。过表达慢病毒感染后HBVSMC有荧光表达,并且荧光率达80%以上,细胞生长良好传后细胞几乎无死亡现象。结论:成功构建了过表达HTRA1及HTRA1-Mut基因的慢病毒表达载体,得到了较高滴度的病毒悬液,建成了稳定表达HTRA1及HTRA1-Mut基因的HBVSMC细胞株,为进一步探讨HTRA1基因及突变后细胞的功能变化提供了良好的研究工具。     

Purification of the Fusicoccin-Binding Protein from Oat Root Plasma Membrane by Affinity Chromatography with Biotinylated Fusicoccin

Fusicoccin (FC), a fungal phytotoxin, evokes a number of physiological responses after binding to the FC-binding protein (FCBP). For characterization of this plasma membrane protein and elucidation of the signal transduction pathway, we purified active FCBP from oat (Avena sativa L. cv Valiant) root plasma membranes using avidin-biotin affinity chromatography. For the binding of FCBP to immobilized avidin, a bifunctional FC derivative (FC-biotin, FCBio) was synthesized. FCBio retained high binding affinity for the FCBP (KD = 70 nM), it elicited a biological response comparable to FC, and it was bound by avidin. The purification of the FCBP involved three important steps. First, FCBio was bound to the FCBP in purified plasma membrane vesicles. Next, plasma membrane proteins were solubilized in detergent, and part of the solubilized proteins was precipitated by decreasing the detergent concentration below the critical micelle concentration. The FCBP remained in the soluble fraction, and this fraction was loaded on a "low-affinity" avidin column. Proteins, bound through a biotin moiety to the column, were specifically eluted with excess biotin. This resulted in fractions active in [3H]FC binding and two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31 and 30 kD. The nonhydrophobic behavior of the FCBP was confirmed by means of phase separation with Triton X-114, wherein the FCBP migrated to the hydrophilic phase. Purification of the FCBP in active form using this novel affinity technique opens the possibility to study other features of the FCBP necessary for inducing physiological responses in plant cells.     

Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.     

用慢病毒载体制备转基因动物的研究进展

慢病毒是逆转录病毒的一种 ,具有逆转录病毒的基本结构 ,但也有不同于逆转录病毒的组份和特性 ,作为基因治疗载体发展起来 ,最近已用于转基因动物制备。慢病毒像其它逆转录病毒一样 ,其基因组经逆转录后能整合在宿主DNA上 ;由于病毒载体经改构后 ,不在宿主细胞增殖 ,不会导致寄主细胞的死亡 ,被它感染的或转化的动物细胞能够连续传代 ;这种载体的最大优势是能感染静止细胞和不产生嵌合体动物。详细介绍了慢病毒载体构建原理、近年慢病毒载体在转基因动物制备方法和应用研究的新进展。     

The purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B

The pH-dependent interaction between the cyclic guanidino analog of biotin, 2-iminobiotin, and avidin has been used in the design of an efficient affinity isolation system for avidin and its fluorescent and iodinated derivatives. Avidin and its derivatives are retained by a column of 2-iminobiotin-6-aminohexyl-Sepharose 4B at pH values between 9 and 11 and are specifically eluted from the column at pH 4. This affinity isolation procedure overcomes the harsh conditions, i.e., 6 m guanidine-HCl, pH 1.5, required to dissociate avidin from an immobilized biotin column.     

Formation of complexes between avidin and β-adrenergic receptors using biotinyl-alprenolol derivatives

The goal of this study was to synthesize biotinylated derivatives of alprenolol, a β-adrenergic antagonist, and to determine whether these ligands could bind simultaneously to both avidin (a biotin-binding protein) and to the β-adrenergic receptor. Such ligands would be useful for β-adrenergic receptor localization and purification, since avidin can be covalently labelled with fluorescent or electron-dense markers or can be linked to solid supports for affinity chromatography. Three biotinyl derivatives of alprenolol were synthesized and characterized. Each derivative bound to avidin and also possesed high affinity for the duck erythrocyte β-adrenergic receptor. Two of the compounds, biotinyl-caproyl-cysteaminyl-alprenolol (BCCA) and biotinyl-dodecanoyl-cysteaminyl-alprenolol (BDCA) had the same affinities for the duck erythrocyte β-adrenergic receptor (membrane-bound or digitonin-solubilized) in the absence and presence of avidin. This indicated that high affinity complexes could be formed between the β-adrenergic receptor and avidin using these bifunctional biotinyl-alprenolol ligands. In contrast, biotinyl-cysteaminyl-alprenolol (BCA), in which the distance between the biotin and alprenolol moieties was shorter, had greatly reduced affinity for the duck erythrocyte β-adrenergic receptor in the presence of avidin. Additional studies showed that BDCA, avidin-BDCA, and ferritin-avidin-BDCA were equally potent in inhibiting the isoproterenol stimulation of cAMP accumulation in intact HeLa cells. The data reported in this paper demonstrate the importance of an appropriate spacer sequence to allow correct apposition of the receptor and avidin molecules, and suggest that BDCA may be a useful probe for β-adrenergic receptor localization and purification.     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Magnetic resonance imaging visualization of targeted cells by the internalization of supramolecular adducts formed between avidin and biotinylated Gd3+ chelates

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin–Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd³⁺ complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6×10⁸ Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T₁ relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.     

Isolation and cultivation of murine hematopoietic stem cells and expression of hFIX mediated by recombinant lentiviral vectors in vitro

Hematopoietic stem cells (HSCs) are an attractive target for gene therapy, especially for inherited blood diseases. Moreover, recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection. In this study, murine mononuclear cells (MNCs) were isolated from bone marrow and cultured in suspension, and then Lin⁻CD117⁺ HSCs were isolated by immunomagnetic beads. During culturing, cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines. FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice. The hFIX expressions were 41.7 ± 4.2 ng / mL and 34.5 ± 6.6 ng/mL in supernatant on 7d. The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6 ± 5.7 ng/mL (with cytokines) and 33.3 ± 4.8 ng/mL (without cytokines) in supernatant on 7d. Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin⁻CD117⁺ HSCs efficiently, and expression of the transgene can be improved when supplied with cytokines. __________ Translated from Journal of Fudan University (Natural Science), 2005, 44(4): 503–506 [译自: 复旦学报(自然科学版), 2005, 44(4): 503–506]     

2′- and 3′- Biotin Conjugated Nucleoside Building Blocks: Synthesis of Biotinylated Oligonucleotides

Abstract

The vitamin biotin plays a significant role in biological assays based on its unusually high affinity [K_D=10^?15M] to streptavidin and avidin. This assay can be used for monitoring cellular trafficking of antisense oligonucleotides using hiotin conjugation. In addition to the above diagnostic application, biotin conjugation to macromolecules could be used as a vitamin-mediated delivery system for macromolecules into cells. Complexation of avidin to hiotin-oligonucleotides (phosphodiesters or PNA) have been used to enhance the uptake of oligonucleotides¹. Appropiiate placement of biotin in oligonucleotides could also provide increased nuclease resistance.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

一种新型慢病毒载体制备体系的初步建立

为了建立新型、高产量的慢病毒载体制备体系,将构建好的主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG通过脂质体共转染至BHK21细胞.再用含有T7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞,培养4天后,收集培养上清.提取培养上清的RNA,进行RT-PCR反应,将培养上清进行免疫印迹鉴定,将培养上清感染正常的293T细胞、HepG2细胞、Vero细胞,荧光显微镜下观察细胞GFP的表达情况,采取3×3×3析因分析方法,优化系统产量,Real-timePCR方法测定细胞培养上清中病毒载体的拷贝数,利用流式细胞术检测病毒载体滴度.RT-PCR及p24免疫印迹结果均提示在细胞上清中存在慢病毒载体;通过荧光显微镜观察到感染组293T细胞、HepG2细胞、Vero细胞均表达绿色荧光蛋白GFP,说明此系统制备出的慢病毒载体具有感染性;系统经优化后,培养上清中慢病毒载体拷贝数达到1.1×1012/ml,培养上清原始滴度达到1.3×108tu/ml,高出目前常用制备体系产量1个数量级.上述结果表明,新型慢病毒载体制备体系已初步建立,为今后该系统的大规模应用提供客观的科学依据.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Vectors derived from simian immunodeficiency virus (SIV)

In contrast to other retroviruses, lentiviruses have the unique property of infecting non-proliferating cells. Thus vectors derived from lentiviruses are promising tools for in vivo gene delivery applications. Vectors derived from human primate and non-primate lentiviruses have recently been described and, unlike retroviral vectors derived from murine leukemia viruses, lead to stable integration of the transgene into quiescent cells in various organs. Despite all the safety safeguards that have been progressively introduced in lentiviral vectors, the clinical acceptance of vectors derived from pathogenic lentiviruses is subject to debate. It is therefore essential to design vectors derived from a wide range of lentivirus types and to comparatively examine their properties in terms of transduction efficiency and bio-safety. Here, we review the properties of lentiviral vectors derived from simian immunodeficiency virus (SIV).