Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.     

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins

In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.     

The utility of affinity-tags for detection of a streptococcal protein from a variety of streptococcal species

There is no systematic examination of affinity tag utility in Gram-positive bacteria, which limits the investigation of protein function in this important group of bacteria as specific antibodies for many of native proteins are generally not available. In this study, we utilized an E. coli-streptococcal shuttle vector pVT1666 and constructed two sets of expression plasmids pVPT-CTag and pVPT-NTag, with each set containing five affinity tags (GST, GFP, HSV, T7 and Nano) that can be fused to either the C- or N-terminus of a target protein. A putative glycosyltransferase (Gtf2) essential for Fap1 glycosylation was used to demonstrate the utility of the cassettes in detection of Gtf2 fusion proteins, and the biological relevance of the proteins in our working strain Streptococcus parasanguinis. GFP and T7 tags were readily expressed in S. parasanguinis as either an N- or C-terminal fusion to Gtf2. Only the C- terminal fusion of GST and HSV were able to be identified in S. parasanguinis. The Nano tag was not detected in either E. coli or S. parasanguinis. Genetic complementation experiments indicated that all the tagged Gtf2 fusion proteins could restore the Gtf2 function in the null mutant except for the Nano-tagged Gtf2 at its N-terminal fusion. Using a T7-tagged Gtf2 fusion construct, we demonstrated that the fusion cassette is also useful in detection of the fusion tag expression in other streptococci including S. mutans, S. pneumoniae and S. sanguinis. Therefore, the expression cassettes we constructed will be a useful tool not only to investigate protein-protein interactions in Fap1 biogenesis in S. parasanguinis, but also to study protein functions in other gram-positive bacteria in which pVT1666 replicates.     

重组GST-HBx蛋白在表达过程中断裂状况的研究

目的 在对乙型肝炎病毒非结构蛋白HBx研究的过程中,发现体外表达HBx蛋白产物的稳定性存在差异.为了了解其中的原因,拟通过原核表达方式进行分析.方法 以包含全长HBV DNA的质粒pEco63为模板,通过PCR反应,扩增HBV X基因全长序列（1～154氨基酸）和截短序列（48～104氨基酸,48～146氨基酸,98～146氨基酸）,将这几种不同长度和位置的HBX DNA序列克隆到pGEX4T-2原核表达载体中,并通过下游引物设计中引入6×组氨酸标签序列.结果 最终成功构建N端融合GST蛋白标签序列和C端融合6×组氨酸标签序列的4种HBx重组表达载体.将4种包含不同长度HBx的重组质粒转化大肠埃希菌Rosetta,培养至菌液浓度A值大约0.6左右,加入终浓度0.1 mmol/L的IPTG进行诱导表达.提取全菌蛋白通过Western印迹对HBx各重组蛋白产物的N端和C端进行检测.结论 在分段表达的HBx重组蛋白中发生了多处局部的断裂,而断裂部位主要集中于GST蛋白上.而造成这一结果的原因可能与HBx蛋白功能区域暴露后与GST相互作用有关.     

Comparison of the efficiencies of different affinity tags in the purification of a recombinant secretory protein expressed in silkworm larval hemolymph

Silkworms are useful bioreactors for heterologous protein expression when used in conjunction with the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. However, purification from silkworm hemolymph is difficult since it contains various kinds of proteins. In this study, we investigated an effective single-step method for the purification of affinity-tagged single-chain antibody variable region fragment (scFv) from silkworm larval hemolymph. A 5-fold higher expression level was obtained when scFv was fused with the His tag than when it was fused with the Strep II or GST tags. However, the His tag was inadequate for single-step purification since it led to the nonspecific binding of contaminants. The purification recoveries of GST-, Strep II-, and His-tagged scFvs were 91.8%, 43.7%, and 27.2%, respectively. The specific amount of single-step purified GST-tagged scFv was 2.2∼2.7 fold higher than the amounts of the His- and Strep II-tagged constructs. The purities of Strep II- and GST-tagged scFvs in the eluent were 98.4% and 83.0%, respectively. Thus, both the short peptide Strep II and GST protein are suitable fusion tags for the affinity purification of proteins from silkworm larvae.     

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.     

Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line.

The cytosolic glutathione transferases (GSTs) with basic pI values have been studied in mouse liver after treatment with 2,3-t-butylhydroxyanisole (BHA), cafestol palmitate (CAF), phenobarbital (PB), 3-methylcholanthrene (3-MC) and trans-stilbene oxide (t-SBO). The cytosolic GST activity was induced by all compounds except for 3-MC. Three forms of GST were isolated by means of affinity chromatography and f.p.l.c. The examination of protein profiles and enzymic activities with specific substrates showed that the three GSTs correspond to those found in control animals, i.e. GSTs MI, MII and MIII. The class Mu GST MIII accounted for the major effect of induction, whereas the class Alpha GST MI and the class Pi GST MII were unchanged or somewhat down-regulated. The greatest induction was obtained with BHA, PB and CAF. The activities of other glutathione-dependent enzymes were also studied. An increase in glutathione reductase and thioltransferase activities was observed after BHA, PB or CAF treatment; glyoxalase I and Se-dependent glutathione peroxidase were depressed in comparison with the control group in all cases studied.     

Fusion with pep-1, a cell-penetrating peptide,enhances the transmembrane ability of human epidermal growth factor

Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.     

Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail.

A method to purify proteins by fusing them to the Ca2+-dependent protein calmodulin is described by using glutathione-S-transferase (GST) from Schistosoma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective factor Xa cleavage site located between the C-terminus of GST and the N-terminus of CaM. The recombinant fusion protein was expressed in Escherichia coli, and the crude cell extract was loaded onto a phenothiazine affinity column in the presence of Ca2+. Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the target protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system

In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification.     

Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain

N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.     

A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system

In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene (PS) surface, a linear 12-mer peptide phage display random library was screened. Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning. The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues, which may be critical to binding. One of the selected dodecapeptides, named Lig1 (FKFWLYEHVIRG), was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1), to investigate its performance as an affinity ligand. The ligand-fused ENVs overexpressed in Escherichia coli were compared to the original one in terms of the immobilization characteristics on PS plate in enzyme-linked immunosorbent assay (ELISA). The results indicated that the ligand-fused proteins showed a considerably improved affinity to PS surface, and were preferentially adsorbed on PS plate suffering only scarcely from interference by coexisting protein molecules. Anti-HIV-1 ELISA system, which employed Lig1-ENV (Lig1 fused to ENV N-terminus) as immobilization antigen also exhibited sufficiently high sensitivity and specificity in serodiagnosis tests.     

Specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.

alpha-, beta-, and gamma-cyclodextrins (CDs) completely inhibited raw starch digestion by glucoamylase I (GA I, MW 90,000) from Aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of GA I at the CD concentrations of 1-5 mM. CDs at 1-5 mM did not inhibit gelatinized starch hydrolysis by GA I, but at the concentration of 50 mM, they inhibited such hydrolysis slightly. GA I was specifically adsorbed onto CD-Sepharose 6B, but glucoamylase I' (GA I', MW 73,000), which does not adsorb onto or digest raw starch, from the same strain was not adsorbed onto that gel. The adsorption of the glucoamylases onto raw starch and CD-Sepharose 6B was correlated to their digestion of raw starch. The hydrophobic adsorption of GA I onto CDs and raw starch occurred competitively at the Cp region, which is on the C-terminal side of Gp-I in the site for raw starch affinity of GA I, and inclusion complexes were formed.     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.     

Expression and binding analysis of GST-VacA fusions reveals that the C-terminal approximately 100-residue segment of exotoxin is crucial for binding in HeLa cells

The Helicobacter pylori toxin VacA induces intracellular vacuolation and plays an essential role in H. pylori-related diseases. The mature exotoxin is divided into two domains, P37 and P58. A soluble form of VacA fused with GST was expressed in Escherichia coli. Although the soluble fusion lacked vacuolating activity after cleavage by thrombin, it had a binding affinity similar to that of the native VacA. Moreover, it blocked the vacuolating activity induced by the native toxin. Different C-terminal truncated fusions were generated (GST-P72, GST-P53, and GST-P37) and were also produced in a soluble form. A significantly reduced binding activity was seen for GST-P72 and nearly no specific association was detected for GST-P37. Our results suggested that the whole P58 fragment contributed to the cell binding activity in HeLa cells, particularly in the C-terminal approximately 100-residue region.