Peptide phage display as a tool for drug discovery: targeting membrane receptors

Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.     

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

Introduction CardiactroponinI(cTnI),aspecificproteinof cardiacmusclecells,showsa40%dissimilarity withskeletaltroponinI(sTnI)inaminoacidse- quence.Moreover,humancardiacTnIhas31addi- tionalresiduesonitsN-terminalend,whichare notpresentinskeletalforms,thusprovidingahigh potentialforobtainingcardiac-specificantibod- ies[1,2].Themolecularweightofthisproteinis29 kDaandtherefore,itwillbereleasedreasonably rapidlyafteracutemyocardialinfarction(AMI). CTnIoftenappearsinbloodwithinafewhoursaf- ter…     

Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development

Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western? (or Simon?) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon? can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.     

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.     

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.     

Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin‐containing poly‐horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP‐secondary antibody instead of HRP‐secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin‐conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin‐conjugated PolyHRP resulted in as much as a 110‐fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin‐conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin‐conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.     

Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins

We used the recombinant phage display antibody system (RPAS) to obtain chimeric single-chain fragment variable (ScFv) antibodies to gill proteins of the white clam Codakia orbicularis (Linné, 1758). After three rounds of selection on immunotubes loaded with total gill protein extract, recombinant phages exhibiting antibodies to gill proteins were isolated and tested by enzyme-linked immunosorbent assay (ELISA). Clones exhibiting a high affinity for the mollusk proteins were selected for production of soluble ScFv antibodies, which were purified for subsequent analysis. ScFv antibodies exhibited a reaction specific for a protein whose molecular mass was about 15,000 Daltons and that was detected by the antigen capture technique followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.     

Functional Mutations in and Characterization of VHH Against Helicobacter pylori Urease

Manipulation of clinically significant antibodies can effectively improve the processes of diagnosis and treatment. Affinity maturation process has a significant role in improvement of antibodies efficiency. Error-prone PCR technique is one of the proposed methods for improvement of the affinity of antibodies. In the present research, a method was applied to camel heavy-chain antibody (VHH, nanobody) raised against UreC subunit of urease enzyme from Helicobacter pylori. This VHH was used as a starting molecule to construct a highly diversified phage displayed VHH library. The constructed library of nanobody mutants was subjected to several rounds of panning against UreC antigen. High-affinity mutant was selected. Our VHH (HMR23) showed 1.5-fold higher binding activity than the parental VHH. In addition, the mutant VHH presented a better performance in inhibition of urease activity at low concentrations retaining its specificity and thermal stability.     

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

IntroductionReactive oxygen species(ROS) are known to de-stroy biomacromolecules and cause cell injury[1]. Un-der normal circumstances, there is a balance betweenthe production of ROS and their destruction. Many dis-eases, such as brain ischemia, tumor, v…     

高密度噬菌体抗体芯片对细胞表面蛋白的识别

采用正常人和白血病患者的白细胞对人源噬菌体抗体库进行淘选, 以获得对两种细胞表面蛋白特异的抗体. 通过pVIII展示系统, 使抗体以多价展示于重组噬菌体颗粒表面, 从上述两组中各挑选出48个克隆分别固定于环氧基片上, 并以空白噬菌体和牛血清白蛋白作为对照, 制成高密度噬菌体抗体芯片. 取来自3名正常人和3名白血病患者的白细胞裂解物样品, 用荧光染料Cy3标记, 与噬菌体抗体芯片反应, 对微阵共聚焦扫描得到的荧光图谱进行分析. 在白血病白细胞表面蛋白的识别图谱中有8组斑点显著不同于正常图谱. 由此表明, 噬菌体抗体芯片可用于识别细胞表面蛋白.     

Heterologous expression of photoactivated adenylyl cyclase (PAC) genes from the flagellate Euglena gracilis in insect cells

The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha (Mr 105 kDa) and two PACbeta (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACalpha and PACbeta were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACbeta and PACbeta, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACalpha and PACbeta antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.     

Cross-reactivity of antibodies to actin- depolymerizing factor/cofilin family proteins and identification of the major epitope recognized by a mammalian actin-depolymerizing factor/cofilin antibody

Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica.     

DNA-encoded antibody libraries: a unified platform for multiplexed cell sorting and detection of genes and proteins

Whether for pathological examination or for fundamental biology studies, different classes of biomaterials and biomolecules are each measured from a different region of a typically heterogeneous tissue sample, thus introducing unavoidable sources of noise that are hard to quantitate. We describe the method of DNA-encoded antibody libraries (DEAL) for spatially multiplexed detection of ssDNAs and proteins as well as for cell sorting, all on the same diagnostic platform. DEAL is based upon the coupling of ssDNA oligomers onto antibodies which are then combined with the biological sample of interest. Spotted DNA arrays, which are found to inhibit biofouling, are utilized to spatially stratify the biomolecules or cells of interest. We demonstrate the DEAL technique for (1) the rapid detection of multiple proteins within a single microfluidic channel, and, with the additional step of electroless amplification of gold-nanoparticle labeled secondary antibodies, we establish a detection limit of 10 fM for the protein IL-2, 150 times more sensitive than the analogue ELISA; (2) the multiplexed, on-chip sorting of both immortalized cell lines and primary immune cells with an efficiency that exceeds surface-confined panning approaches; and (3) the co-detection of ssDNAs, proteins, and cell populations on the same platform.     

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.     

Preparation and Application of Monoclonal Antibody Against hNDRG2

The full-length hNdrg2 cDNA-coded 357 amino acids was cloned and expressed in Escherichia coli strain DH5α as a 6× His-tagged protein. The purified 6× His-fusion protein was used to immunize mice for preparing monoclonal antibodies (mAb) against N-myc downstream-regulated gene 2 (Ndrg2). A hybridoma secreting a monoclonal antibody against Ndrg2 was obtained and named FMU-Ndrg2.3. Western blot analysis confirmed that this mAb is specific only to Ndrg2 but not to Ndrg1, Ndrg3, and Ndrg4-B. Some tissue distribution features of Ndrg2 proteins, such as thyroid, kidney, testis, prostate, and pancreas islets, were present by immunohistochemistry.     

A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB

Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low‐abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β‐mercaptoethanol, SDS, and Tris‐HCl and a washing buffer consisting of PBS added with 0.1% Tween‐20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.     

Increase in expression of P2X1 receptors in the atria of patients suffering from dilated cardiomyopathy

P2X1 receptors are ATP-sensitive ligand-gated cation-selective channels abundant in smooth muscle tissues such as bladder and vas deferens. They have also been detected in the central and peripheral nervous system and in heart tissue. We have earlier reported distinct changes in the expression of the PX1 subtype of P2X receptors in hearts of patients suffering from dilated cardiomyopathy (DCM). The study was, however, based on Western blots from only five DCM samples and three control hearts. Moreover, the antibody was directed against a peptide derived from the sequence of rat P2X1. In the present project we have examined larger groups of both DCM and control hearts (n = 14 and 11, respectively). Furthermore, the antibody used in this paper differs significantly from the one used in our previous report. The present antibody was raised against an 18-residue peptide sequence (Lys 68-84 Val) derived from the human P2X1 sequence. Most of the label in the Western blots was concentrated over a triplet of bands migrating with an apparent Mr of about 45,000. Quantitative densitometry indicated that this band was more strongly expressed (by approximately 80%) in DCM hearts compared with the controls.     

Electrophoretic separation of ubiquitin and single amino acid residue ubiquitin extensions using a commercial modified acrylamide gel electrophoresis system: an assay to determine catalytic capacities of deubiquitinating enzymes

We have chemically synthesised a number of ubiquitin extension proteins, with carboxyl-terminal single amino acid residue extensions, to use as substrates to assess the catalytic capacities of deubiquitinating enzymes (DUBs). Here we describe a modified acrylamide gel electrophoresis system which allows separation of peptide- or isopeptide-linked ubiquitin-lysine from ubiquitin (77 and 76 residue proteins respectively) in only 2 h. Western blotting, using antibodies against ubiquitin, allows both substrate (i.e. ubiquitin-lysine) and product (i.e. ubiquitin) of DUB-catalyzed cleavage reactions to be detected. Catalytic capacities of DUBs may be indicative of in vivo functions of these proteases.     

DNA and RNA aptamers: from tools for basic research towards therapeutic applications

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.