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.     

Anchor-chain molecular system for orientation control in enzyme immobilization

An anchor-chain molecular system was constructed for controlled orientation and high activity in enzyme immobilization. A streptavidin recognition peptide (streptag) coding sequence was fused to the 3' end of the phoA gene, which codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT) and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequence with or without the insertion of a flexible linker peptide [-(Gly-Ser)(5)-] coding sequence. The fused genes were cloned into the vector pASK75 and expressed in the periplasm of the host cell Escherichia coli SM547. The proteins were released by osmotic shock and purified by ion-exchange chromatography. Enzyme activities of all proteins were measured spectrophotometrically with rho-nitrophenyl phosphate as the substrate. Specific activities of D101S-streptag and D101S-linker-streptag enzymes were increased 25- or 34-fold over the WT, respectively. These fusion proteins were then immobilized on microtiter plates through streptag-streptavidin binding reaction. After immobilization, the D101S-linker-streptag enzyme displayed the highest residual activity and the ratio of enzyme activities of the linker to nonlinker enzymes was 8.4. These results show that the addition of a linker peptide provides a spacer so as to minimize steric hindrance between the enzyme and streptavidin. The method provides a solution for controlled enzyme immobilization with high recover activity, which is especially important in construction of biosensors, biochips, or other biodevices.     

Fluorogen-Activating scFv Biosensors Target Surface Markers on Live Cells Via Streptavidin or Single-Chain Avidin

Fluorescence biosensors are indispensable tools for understanding protein behavior and function in cells. Recent advancements utilize fluorogen-activating-proteins (FAPs) that form complexes with small organic molecules (fluorogens) and result in their fluorescence activation. The technology has found multiple uses in protein discovery applications; however, the current method of detection requires the expression of FAPs as gene fusion tags in cells—a process that is time- and labor-intensive. In this report, we present an alternate method that utilizes FAPs as affinity reagents. Accordingly, we isolated soluble reagents based on FAP fusions with streptavidin (Strep) or avidin proteins, both highly selective for biotin. When tested in vitro, the reagents displayed bi-functional activity, fluorogen activation, and biotin affinity. For live-cell protein discovery, surface targets were biotinylated via biotin-tagged immunoglobulins or a genetically encoded biotin acceptor peptide. As a result, when the cells were labeled with FAP–Strep or FAP–avidin reagent, the in vivo fluorescence measurements indicated high target specificity, minimal background, and bright signal detection. In summary, we present a novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization,yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.     

A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.     

High avidity binding of engineered papaya mosaic virus virus-like particles to resting spores of Plasmodiophora brassicae

Papaya mosaic virus (PapMV) like particles (VLPs) were used as a platform for fusion of affinity peptides binding to resting spores of Plasmodiophora brassicae-a major pathogen of crucifers. Three peptides with specific affinity to the target were isolated and cloned at the C-terminus of the PapMV coat protein (CP), generating three different high avidity VLPs. The peptides were exposed at the surface of the VLPs and their avidity to resting spores of P. brassicae was measured by flow cytometry. NLP-A, with the peptide DPAPRPR, showed the highest avidity. The binding avidity of NLP-A to P. brassicae spores was comparable to that of a polyclonal antibody. NLP-A was also shown to be more specific than the antibody. Fusion of the affinity peptide to a monomeric form (mCP) of the CP [Lecours, K., Tremblay, M.-H., Laliberté Gagné, M.-E., Gagné, S.M., Leclerc, D., 2006. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein. Protein Express. Purific. 47, 273-280] generated a fusion protein that was unable to assemble into VLPs, and mCP-A fusions failed to bind resting spores. The avidity of VLP-A was increased by adding a glycine spacer between the C-terminus of the PapMV CP and the peptide, and improved even further by using a duplicated A peptide in the fusion protein. The use of high avidity VLPs has advantages over polyclonal antibodies because of target specificity. VLPs offers the specificity of monoclonal antibodies but can be more easily generated using the powerful selection of phage display.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

Production of recombinant peptides as fusions with SUMO

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.     

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.     

The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide – instead of D-biotin recognized by the natural protein – to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.     

Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein,I-Au,which confers susceptibility to experimental allergic encephalomyelitis.

BACKGROUND: An important issue in autoimmune diseases mediated by T cells, such as experimental allergic encephalomyelitis (EAE), is the affinity of the disease-inducing determinants for MHC class II proteins. Tolerance, either due to clonal deletion or anergy induction, is thought to require high-affinity interactions between peptides and MHC molecules. Low-affinity binding is compatible with the hypothesis that breaking tolerance to self proteins does not have to occur for onset of disease. In contrast, a high-affinity interaction implies that an event leading to a breakdown of tolerance is central to the autoimmune process. MATERIALS AND METHODS: Detergent-solubilized and affinity-purified I-Au was incubated with varying concentrations of a set of peptides from myelin basic protein and a biotinylated peptide agonist. The specific complexes were separated from excess peptide by capture on antibody-coated plates, and the affinity of the peptides was measured by adding europium-labeled streptavidin and measuring the resultant fluorescence. RESULTS: The immunodominant and encephalitogenic determinant, Ac 1-11, was shown to bind to I-Au relatively poorly (IC50 = 100 microM), demonstrating that in this protein, immunodominance did not correlate with high-affinity binding. In contrast with the natural sequence, the ability of shorter analogs to induce EAE did correlate with their apparent affinity. CONCLUSIONS: The dominance of the natural determinant does not arise from a high-affinity interaction with the MHC class II molecule. This suggests that other mechanisms are operative and that the specific T cell for this peptide/MHC ligand is of high affinity.     

Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.     

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.     

Escherichia coli peptide binding protein OppA has a preference for positively charged peptides

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.     

A Technology for Developing Synbodies with Antibacterial Activity

The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.     

Design of a highly potent inhibitory peptide acting as a competitive inhibitor of HMG-CoA reductase

This study presents a design of a highly potent and competitive inhibitory peptide for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). HMGR is the major regulatory enzyme of cholesterol biosynthesis and the target enzyme of many investigations aimed at lowering the rate of cholesterol biosynthesis. In previous studies, the two hypocholesterolemic peptides (LPYP and IAVPGEVA) were isolated and identified from soy protein. Based on these peptide sequences, a number of peptides were designed previously by using the correlation between the conformational flexibility and bioactivity. The design method that was applied in previous studies was slightly modified for the purpose of the current research and 12 new peptides were designed and synthesized. Among all peptides, SFGYVAE showed the highest ability to inhibit HMGR. A kinetic analysis revealed that this peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K _i) of 12?±?0.4?nM. This is an overall 14,500-fold increase in inhibitory activity compared to the first isolated LPYP peptide from soybeans. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide

The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.