Quantifying beta-sheet stability by phage display

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.     

Selection of silk-binding peptides by phage display

Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications.     

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.     

噬菌体展示技术筛选bFGF模拟短肽

目的：通过噬菌体展示技术筛选得到与FGFR结合的bFGF模拟短肽,为bFGF肽类抑制剂的研发提供实验基础。方法：以Balb/c 3T3细胞为靶标,以COS-7细胞作消减,对噬菌体随机七肽库进行4轮生物淘洗,再采用ELISA检测单克隆噬菌体对Balb/c 3T3亲和性和特异性,选取阳性克隆进行DNA测序分析。结果：从富集的噬菌体中获得12个阳性克隆,获得一组疏水性七肽及共同基序PR。结论：利用肽类新药开发的重要工具--噬菌体展示技术,得到2段bFGF的受体结合模拟肽,可望作为bFGF抑制剂的先导肽。     

Selection of HBV preSl-binding penta-peptides by phage display

Chronic hepatitis B virus （HBV） infection can lead to liver cirrhosis and hepatocellular carcinoma. Current therapies have a very limited efficacy in virus clearance. New anti- viral targets and agents are urgently needed. The envelope of HBV virion contains three surface glycoproteins, namely the large （LHBs）, middle （MHBs）, and small （SHBs） pro- teins. LHBs has an amino terminal preS which is composed of the preS1 and preS2 domains. The amino half of preS1 which is myristoylated plays a pivotal role in HBV entry, which can be exploited as an antiviral target. A common motif of five amino acids had been previously discovered to bind preSll_~s and HBV particles. In this study, we used preSl 1-65 to screen a phage display library of random penta-peptides to select the penta-peptides possessing a high preSl-binding affinity. After nine rounds of panning, we obtained one peptide designated as A5 which could bind preS1 with a high affinity. By systematically substitut- ing each residue of A5 with the other 19 amino acids, we identified a novel peptide with an increased preSl-binding affinity. Both peptides could inhibit HBV attachment to HepG2 cells, making them be potential candidates for HBV entry inhibitors.     

Selection of a buried salt bridge by phage display

The α-helical coiled coil is a valuable folding motif for protein design and engineering. By means of phage display technology, we selected a capable binding partner for one strand of a coiled coil bearing a charged amino acid in a central hydrophobic core position. This procedure resulted in a novel coiled coil pair featuring an opposed Glu-Lys pair arranged staggered within the hydrophobic core of a coiled coil structure. Structural investigation of the selected coiled coil dimer by CD spectroscopy and MD simulations suggest that a buried salt bridge within the hydrophobic core enables the specific dimerization of two peptides.     

Selection of Burkholderia pseudomallei protease-binding peptides by phage display

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. A protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We have used phage display technology to identify peptides binding to B. pseudomallei protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The cyclic heptapeptides from two of the phage clones (Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys) were identical and showed the strongest phage-protease interaction as detected by ELISA. Four of the five selected phages at the amount of 10¹³ phages could inhibit B. pseudomallei protease activity by approximately 50%.     

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.     

An improved phage display procedure for identification of lipopolysaccharide-binding peptides

We successfully implemented several modifications to the regular phage display procedure and significantly improved the lipopolysaccharides-binding properties of the peptides selected. Specifically, the number of biopannings was increased and peptides with consensus sequences were obtained. A dual selection procedure (referred to as subtractive panning) was used to simultaneously select for the desired target and deselect for an undesired target, thereby increasing the binding specificity. In addition, binding and washing conditions in the subtractive panning were also modified to favor the selection of peptides with higher binding strength. As a result, two peptides, ASFPPAF and SSHTISF, were identified with much improved binding properties compared to those selected with regular panning. The binding specificities of these two peptides, as measured by the ratio of phages bound to the desired and undesired targets, were severalfold higher than previously reported. These modifications could easily be implemented with many other target molecules, indicating the general applicability of the procedure.     

Selection of large diversities of antiidiotypic antibody fragments by phage display

Antiidiotypic antibodies (Ab2) are needed as tools for a better understanding of molecular mimicry and the immunological network, and for many potential applications in the biomedical and pharmaceutical field. Antiidiotypic antibodies mimicking carbohydrate or conformational epitopes (Ab2beta) are of considerable interest as surrogate immunogens for cancer vaccination. However, it has so far been difficult and tedious to produce Ab2s to a given antigen. Here we describe a fast and reliable technique for generating large diversities of antiidiotypic single chain antibody fragments from non-immunized phagemid libraries using phage display. Key elements are a specific elution with the original antigen followed by trypsin treatment of the eluted phages in combination with the protease sensitive helperphage KM13. This novel method was compared with various conventional selection and elution methods, including, specific elution with or without trypsin treatment, elution with glycine at pH 2.2 with or without trypsin treatment, and elution by trypsin treatment only. The results clearly show that specific elution in combination with trypsin treatment of the eluted phages is by far superior to the other conventional methods, enabling for the first time the generation of a large variety of Ab2s after only two to three rounds of selection, thereby maintaining maximum diversity. We obtained 28 to 88 antiidiotypes out of 96 tested clones after two to three rounds of selection with a diversity of 55-90 %. This was achieved for two carbohydrate (di-, and tetrasaccharides) and one conformational protein epitope using two large na?ve libraries and their corresponding monoclonal Ab1. The antiidiotypic nature of the selected scFv-phages was verified by ELISA and immunocytochemistry inhibition experiments.     

An improved helper phage system for efficient isolation of specific antibody molecules in phage display

Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F⁺ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.     

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology.     

Selection of biologically active peptides by phage display of random peptide libraries

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein—protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.     

Selection and characterization of human monoclonal antibodies against Abrin by phage display

Abrin is a highly potent and lethal type II ribosome inactivating toxin that may be used as a biological warfare agent. To date, no human anti-Abrin antibodies have yet to be reported. Herein, we describe the selection and characterization of two human monoclonal antibodies, termed E12 and RF12, which are capable of binding native Abrin with high affinity and specificity. Through surface plasmon resonance studies, we have determined the association and dissociation rate constants and the cross-reactivity for both antibodies. In our developed Biacore-based Abrin detection system, the limit of detection of antibodies E12 and RF12 is 35 and 75 ng/mL, respectively. These concentrations are about 5 x 10(4)-fold lower than the extrapolated Abrin human LD(50). In sum, our data demonstrated the power of human antibody phage display libraries and the promise of these antibodies as detection devices for Abrin.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

Functional cloning by phage display

This review focusses on the isolation of proteins from genomic or cDNA expression products libraries displayed on phage. The use of phage display is highlighted for the characterization of binding proteins with diverse biological functions. Phage display is compared with another strategy, the yeast two-hybrid method. The combination of both strategies is especially powerful to eliminate false positives and to get information on the biochemical functions of proteins.     

Transdermal protein delivery by a coadministered peptide identified via phage display

Efficient transdermal drug delivery of large hydrophilic drugs is challenging. Here we report that the short synthetic peptide, ACSSSPSKHCG, identified by in vivo phage display, facilitated efficient transdermal protein drug delivery through intact skin. Coadministration of the peptide and insulin to the abdominal skin of diabetic rats resulted in elevated systemic levels of insulin and suppressed serum glucose levels for at least 11 h. Significant systemic bioavailability of human growth hormone was also achieved when topically coadministered with the peptide. The transdermal-enhancing activity of the peptide was sequence specific and dose dependent, did not involve direct interaction with insulin and enabled penetration of insulin into hair follicles beyond a depth of 600 microm. Time-lapse studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular drugs to reach systemic circulation.