Expression, refolding and indirect immobilization of horseradish peroxidase (HRP) to cellulose via a phage-selected peptide and cellulose-binding domain (CBD).

We examined the potential immobilization of horseradish peroxidase (HRP) to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library. A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form. The sequence I/LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag. The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose via three amino acids selected by phage display library and CBD.     

Identification of a polymeric β-cyclodextrin-binding peptide from a phage-displayed peptide library

In this work, a phage-displayed peptide library was applied to identification of β-cyclodextrin (CD)-binding peptide tag, capable of being combined to target peptides or proteins in a homogeneous way by established methods such as peptide synthesis and the recombinant DNA technique. Four enriched sequences were obtained after five rounds of biopanning against polymeric β-CD beads. One of the sequences showed high binding affinity to β-CD beads with a dissociation constant of approximately 7 × 10^–6 M. The β-CD-binding sequence was used for immobilization of a hepatitis C virus (HCV) antigenic peptide on β-CD beads. The functionalized β-CD beads were successfully used for immunoassay of anti-HCV antibody with a detection limit of 1 ng. These results demonstrate that the identified peptide sequence has the potential of being used as an affinity tag to β-CD-containing surfaces.     

mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae

General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.     

Discovery of thioether-bridged cyclic pentapeptides binding to Grb2-SH2 domain with high affinity

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low micromolar range binding affinity, such as cyclic peptide 15 with an K_d = 0.359 μM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.     

A screening of phage displayed peptides for the recognition of fullerene (C60)

A novel approach to develop a peptide, that can recognize fullerene (C60) is described for affinity selection of phage displayed peptides from a combinatorial peptide library. Biopanning was performed using cyclic 7-mer peptide library against C60 films deposited on silicon (Si) substrate, and eluted phages with organic solvent. The phage, that recognized C60 films deposited on Si substrate, were obtained from biopanning. The nucleotides of the phage, coding a cyclic 7-mer peptide, were sequenced by standard method. Seventeen kinds of peptide displayed phages were obtained. One kind of peptide (peptide No. 4) displayed phage recognized the C60 films deposited on Si substrate. Peptide No. 4 displayed no affinity towards the Si substrate. The recognition event was monitored by a fluorescent immunoassay. Additionally, peptide No. 4 phage could recognize C60 in powder form, but not the graphite powder. This recognition event in powder form was also observed by a fluorescent immunoassay.     

Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display

We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.     

Tandem affinity purification in Drosophila: the advantages of the GS-TAP system

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.     

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa‐histidine tag, all attached to the C‐terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C‐ or N‐terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His₆ tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase

A Ca2+ -dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+ -ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 amino acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4- to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general.     

Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization

We have exploited emulsion-based in vitro compartmentalization (IVC) to devise a method for the selection of zinc finger proteins (ZFPs) on the basis of their DNA-binding specificity. A library of ZFPs fused to a C-terminal peptide tag is encoded by a set of DNA cassettes that are prepared wholly in vitro. In addition to the ZFP gene, each DNA cassette also carries a given DNA target binding site sequence for which one wishes to isolate ZFP binders. An aliquot of the library is added to bacterial S30 extract and emulsified in mineral oil so that most of the aqueous droplets contain, on average, no more than one gene. If an intra-compartmentally expressed ZFP binds specifically to its encoding DNA via the target binding site, the complex can be purified by affinity capture via the peptide tag after breaking the emulsion, thus rescuing the gene. We present proof-of-principle for this IVC selection method by selecting a specific high-affinity ZFP gene from a high background of a related gene. We also propose that high-affinity ZFPs can be used as genotype-phenotype linkages to enable selection of other proteins using IVC.     

Periplasmic production via the pET expression system of soluble,bioactive human growth hormone

A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni²⁺ affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.     

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.     

The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins

The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.     

The use of a proteinaceous “cushion” with a polystyrene‐binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a “cushion” between the PS‐affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS‐affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527–534, 2016     

Development of a fluorescent cardiomyocyte specific binding probe

Cardiomyocytes are the major component of the heart. Their dysfunction or damage could lead to serious cardiovascular diseases, which have claimed numerous lives around the world. A molecule able to recognize cardiomyocytes would have significant value in diagnosis and treatment. Recently a novel peptide termed myocyte targeting peptide (MTP), with three residues of a non-natural amino acid biphenylalanine (Bip), showed good affinity to cardiomyocytes. Its selectivity towards cardiac tissues was concluded to be due to the ability of Bip to bind cardiac troponin I. With the aim of optimizing the affinity and the specificity towards cardiac myocytes and to better understand structure–activity relationship, a library of MTP derivatives was designed. Exploiting a fluorescent tag, the selectivity of the MTP analogs to myocardium over skeletal and stomach muscle tissues was assayed by fluorescence imaging. Among the tested sequences, the peptide probe Bip2, H-Lys(FITC)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Ser-Gly-Ser-Bip-Bip-NH₂, displayed the best selectivity for cardiomyocytes.     

Use of a novel affinity tag selected with a bacterial random peptide library for improving activity retention of glutathione S-transferase adsorbed on a polystyrene surface

Aiming at developing a novel affinity tag for site-specific immobilization of functional proteins onto polystyrene (PS) surfaces, Escherichia coli random peptide display library was screened for dodecapeptides exhibiting a high affinity toward PS plates. The selected peptides were commonly rich in hydrophobic amino acid residues and had two or three basic amino acid residues. Adsorption and desorption experiments for one of the selected peptide named PS1 (KGLRGWREMISL) showed that it was well and irreversibly adsorbed onto PS latex particles. To study its performance as an affinity tag, PS1 was genetically fused to a model enzyme, glutathione S-transferase (GST), in several manners, and the fusion enzymes were compared to the original GST in terms of the adsorption behavior onto the PS latex particles as well as the specific activities before and after the adsorption. The fusion GSTs in solution showed lower specific activities than the original one, and their adsorption behaviors were also altered. In particular, the fusion of PS1 to the N-terminal region of GST resulted in severe losses both in the specific activity and in the adsorptive ability. However, two types of GSTs fused with PS1 at the C-terminal region were well adsorbed onto the PS latex particles, and their specific activities after the adsorption were significantly higher than the original GST adsorbed on the PS latex particles. The fusion of PS1 to the C-terminal region of GST was thus shown to reduce the activity loss upon the adsorption onto the PS latex particles.     

Protein expression strategies for identification of novel target proteins

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.