Construction of a gene of the human tumor-associated antigen VNTR(MUC1) bound to streptavidin, its expression inEscherichia coli, and the study of properties of the hybrid protein

A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed. It was shown that the streptavidin leader peptide ensures effective secretion of the hybrid protein into the periplasmic space ofEscherichia coli cells. The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.     

Secretion of streptavidin from Bacillus subtilis.

Streptavidin is an extracellular tetrameric protein produced by Streptomyces avidinii. A series of hybrid gene fusions consisting of Bacillus signal peptide coding regions fused to the mature streptavidin sequence were constructed. B. subtilis strains harboring these plasmids accumulate a tetrameric streptavidin in the growth medium. The properties of the streptavidin produced by B. subtilis are similar to those of the streptavidin produced by S. avidinii. B. subtilis strains carrying the various fusions can be grown to a high cell density in a biotin-free medium. Thus, B. subtilis represents an alternate host system for the production of streptavidin.     

Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates.

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.     

Cloning of streptavidin gene from Streptomyces avidinii and its expression in Escherichia coli. Secretion of streptavidin by E. coli cells]

The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created. It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells. The degradation site of the leader peptide was detected. Upon treatment with the total fraction of proteases secreted by S. avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.     

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。     

Genetic engineering of streptavidin,a versatile affinity tag

Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of streptavidin and to expand the application of streptavidin as an affinity tag. In this review, we describe several genetically engineered streptavidin variants, which include a streptavidin with a reduced biotin-binding affinity, a dimeric streptavidin, and a fusion protein between streptavidin and protein A, along with their potential applications in biological science.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.     

Reduced antibody response to streptavidin through site-directed mutagenesis

Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs

DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.     

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.     

The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins

We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag(15) and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag(9) revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate.     

Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

Chimeric avidin shows stability against harsh chemical conditions--biochemical analysis and 3D structure

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.     

A favorable solubility partner for the recombinant expression of streptavidin

Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E. coli as a solubility partner. Domain I (residue 1-158) of IF2 is superior to the well-established solubility partners maltose-binding protein (MBP) and NusA for soluble expression of active streptavidin. The number of active streptavidin molecules isolated by chromatography is increased threefold when domain I is used as solubility partner as compared to MBP or NusA. The relatively small size, high expressivity, and extreme solubility make domain I of IF2 an ideal partner for streptavidin and may also prevent other recombinant proteins such as ScFv antibodies from being expressed as insoluble aggregates in the cytoplasm of E. coli.     

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.