Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Screening of large molecule diversities by phage display

Molecules with tailored binding specificities are needed for many purposes such as the development of therapeutics, the detection and purification of biomolecules or in chemical biology for the study and manipulation of biological systems. With phage display technology, polypeptides with binding affinities to targets of interest can be isolated from billions of polypeptide variants with a modest amount of effort, time and cost. The technology was initially used for the generation and screening of peptide and antibody libraries and was later applied to many different protein scaffolds. More recently, chemically and structurally diverse molecule libraries were generated by chemically modifying phage-displayed polypeptides. In this article, the different classes of natural and non-natural structures that can be encoded and screened by phage display are reviewed with a special focus on bicyclic peptides that we routinely generate in our laboratory.     

Diversity and censoring of landscape phage libraries

Libraries of random peptides displayed on the surface of filamentousphages are a valuable source for biospecific ligands. However,their successful use can be hindered by a disproportionate representationof different phage clones and fluctuation of their compositionthat arises during phage reproduction, which have potentialto affect efficiency of selection of clones with an optimalbinding. Therefore, there is a need to develop phage displaylibraries with extended and varied repertoires of displayedpeptides. In this work, we compared the complexity, evolutionand representation of two phage display libraries displayingforeign octamers and nonamers in 4000 copies as the N-terminalpart of the major coat protein pVIII of phage fd–tet (landscapelibraries). They were obtained by replacement of amino acids2–4 and 2–5 of pVIII with random octa- and nonamers,respectively. Statistical analysis of the libraries revealedtheir dramatic censoring and evolution during amplification.Further, a survey of both libraries for clones that bind commonselectors revealed the presence of different non-overlappingfamilies of target-specific clones in each library justifyingthe concept that different landscape libraries cover differentareas of a sequence space.     

Efficient production of antibodies against a mammalian integral membrane protein by phage display

The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity. We report here a method that can be easily and generally applied to sorting of phage display libraries with multispan protein targets solubilized in detergent. A key feature of this approach is the production of biotinylated multispan proteins in virions of a baculovirus vector that allows library panning without prior purification of the target protein. We obtained Fab fragments from a na?ve synthetic antibody phage library that, when engineered into full-length immunoglobulin (Ig)G, specifically bind cells expressing claudin-1, a protein with four transmembrane regions that is used as an entry co-receptor by the hepatitis C virus (HCV). Affinity-matured variants of one of these antibodies efficiently inhibited HCV infection. The use of baculovirus particles as a source of mammalian multispan protein facilitates the application of phage display to this difficult class of proteins.     

A carbohydrate binding module as a diversity-carrying scaffold

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.     

Exploring protein-protein interactions with phage display

Protein–protein interactions mediate essentially all biological processes. A detailed understanding of these interactions is thus a major goal of modern biological chemistry. In recent years, genome sequencing efforts have revealed tens of thousands of novel genes, but the benefits of genome sequences will only be realized if these data can be translated to the level of protein function. While genome databases offer tremendous opportunities to expand our knowledge of protein–protein interactions, they also present formidable challenges to traditional protein chemistry methods. Indeed, it has become apparent that efficient analysis of proteins on a proteome‐wide scale will require the use of rapid combinatorial approaches. In this regard, phage display is an established combinatorial technology that is likely to play an even greater role in the future of biology. This article reviews recent applications of phage display to the analysis of protein–protein interactions. With combinatorial mutagenesis strategies, it is now possible to rapidly map the binding energetics at protein–protein interfaces through statistical analysis of phage‐displayed protein libraries. In addition, naïve phage‐displayed peptide libraries can be used to obtain small peptide ligands to essentially any protein of interest, and in many cases, these binding peptides act as antagonists or even agonists of natural protein functions. These methods are accelerating the pace of research by enabling the study of complex protein–protein interactions with simple molecular biology methods. With further optimization and automation, it may soon be possible to study hundreds of different proteins in parallel with efforts comparable to those currently expended on the analysis of individual proteins.     

Antibody phage display libraries: contributions to oncology

Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I-III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.     

Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.     

Landscape phages and their fusion proteins targeted to breast cancer cells

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.     

Selection of an active enzyme by phage display on the basis of the enzyme's catalytic activity in vivo

We have developed a novel phage display method based on catalytic activity for the in vivo selection of an enzyme. To confirm the validity of our method and to demonstrate its potential utility, we used biotin protein ligase (BPL) from Escherichia coli as a model enzyme. We were able to demonstrate the potential value of our method by selective enrichment for the birA gene, which encodes BPL, in a mixed library. The presented method for in vivo selection should allow selection of various enzymes that catalyze modification of peptides or proteins, such as protein ligase, acetylase, kinase, phosphatase, ubiquitinase, and protease (including caspase). The method should be useful in efforts to analyze mechanisms of signal transduction, to find unidentified enzymes encoded by cDNA libraries, and to exploit artificial enzymes.     

Phages and HIV-1: From Display to Interplay

The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures.     

Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage

Phage display of antibody libraries has been widely used for over a decade to generate monoclonal antibodies. Yeast display has been developed more recently. Here the two approaches were directly compared using the same HIV-1 immune scFv cDNA library expressed in phage and yeast display vectors and using the same selecting antigen (HIV-1 gp120). Yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting all the scFv identified by phage display and twice as many novel antibodies. Positive phage display selection appeared to largely reflect those antibodies that as phage-scFv gave the highest signal in phage ELISAs assessing antigen binding. This signal is thought to reflect the efficiency of expression of folded scFv at the phage surface. Increased access to immune repertoires may increase the rescue of novel antibodies of therapeutic or analytical value that often form a minor part of a typical antibody response.     

Combinatorial strategies for targeting protein families: application to the proteases

Tens of thousands of proteins have been identified as a result of recent large scale genomic and proteomic efforts. With this large influx of new proteins, the formidable task of elucidating their function begins. However, this task becomes more manageable if proteins are divided into families based upon sequence homology, thereby allowing tools for their systematic study to be developed based upon their common structural and mechanistic characteristics. Combinatorial chemistry is ideally suited for the systematic study of protein families because a large amount of diversity can be readily displayed about a common scaffold designed to target a given protein family. Targeted combinatorial libraries have been particularly effective for the study of a ubiquitous family of proteins, the proteases. Substrate-specificity profiles of many proteases have been determined by using combinatorial libraries of appropriately labeled peptides. This specificity information been utilized to identify the physiological protein substrates of these enzymes and has facilitated inhibitor design efforts. Furthermore, combinatorial libraries of small molecules prepared with mechanism-based scaffolds have resulted in the identification of potent, small-molecule inhibitors of numerous proteases. Cell-permeable small-molecule inhibitors identified by these methods have served as powerful chemical tools to study protease function in vitro and in vivo and have served as leads for the development of therapeutic agents.     

生物支架系统在合成生物学中的应用

合成生物学作为一种新兴的工程化生物学,可以在底盘细胞中引入外源基因模块实现新功能。但是如何将多种外源模块（酶）进行有效地组装从而提高其协同催化功能是急需解决的一个重大问题。由于异源酶在新宿主中存在内源环境适应性问题,限制了酶的生物活性。生物支架系统作为一种有效手段,可以提供有效的多酶组装系统。恰当的生物支架能够提供适应环境的柔性平台,实现多酶体系的表达调控,稳定性组装,利于酶与底物结合的区域化设计。本综述对不同类型的生物支架的研究进展进行了系统的总结。文中根据不同类型支架（蛋白型、核酸型）的特点,介绍了典型的应用范例,论述了每种支架的优势与不足,并对生物支架的常见工作机制做了详细讨论。最后对生物支架在人工细胞器设计和复杂聚合物降解等方面的应用做出展望。     

High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.     

Ribosome display of mammalian receptor domains

Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology.     

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.