A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.     

Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.     

The PASTA server for protein aggregation prediction

Many different proteins aggregate into amyloid fibrils characterized by cross-beta structure. beta-strands contributed by distinct protein molecules are generally found in a parallel in-register alignment. Here, we describe the web server for a novel algorithm, prediction of amyloid structure aggregation (PASTA), to predict the most aggregation-prone portions and the corresponding beta-strand inter-molecular pairing for a given input sequence. PASTA was previously shown to yield results in excellent agreement with available experimental observations, when tested on both natively unfolded and structured proteins. The web server and downloadable source code are freely accessible from the URL: http://protein.cribi.unipd.it/pasta/.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important forcatalysis are poorly understood. We have probed the functionof interdomain interactions by generating tPA variants in whichdomains are duplicated or rearranged. The proteins were expressedin a transient mammalian expression system and tested in vitrofor their ability to activate plasminogen, induce fibrinolysisand bind to a forming fibrin clot. Duplication of the heavychain domains of tPA produced enzymatically active tPA variants,many of which demonstrated similar in vitro amidolytic and fibrinolyticactivity and similar fibrin affinity to the parent molecule.Zymographic analysis of the domain duplication tPA variantsshowed one major active species for each variant. Selectionof the residues duplicated and the interdomain spacing werefound to be critical considerations in the design of tPA variantswith duplicated domains. We also rearranged the domains of tPAsuch that kringle 1 replaced the second kringle domain and viceversa. An analysis of these variants indicates that the firstkringle domain can confer fibrin affinity to a tPA variant andfunction in place of kringle 2. Therefore, in wild-type tPA,the functions of kringle 1 and kringle 2 must be dependent partiallyon their orientation within the heavy chain of the protein.The functional autonomy of the heavy and light chains of tPAis demonstrated by the activity of a tPA variant in which theorder of the heavy and light chains was reversed.     

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.     

An improved method for an efficient and easily accessible eukaryotic ribosome display technology

Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. However, this technology has so far been perceived as being technically challenging owing to comparatively difficult protocols and the absence of tailored commercial reagents, particularly when using prokaryotic cell-free expression systems. Eukaryotic ribosome display is potentially a more accessible alternative because of the availability of suitable commercial reagents, yet despite published protocols, this method has been less widely used. For eukaryotic ribosome display, a novel mechanism of mRNA recovery compared with that of the well-proven prokaryotic method has been proposed. We have examined the eukaryotic ribosome display process with the aims of investigating the proposed mechanism of sequence recovery and of identifying aspects of the protocol that may have lead to poor performance and therefore so far limited its use. We demonstrate that the proposed novel method is in fact mechanistically comparable to the prokaryotic method and we provide a step-by-step protocol for eukaryotic ribosome display that is 20-fold more efficient than current published methods. Our findings should increase the ease of operating ribosome display technology, making it more accessible to the scientific community.     

Selection of single domain binding proteins by covalent DNA display

Selection technologies such as phage and ribosome display, which provide a physical linkage between genetic information and encoded polypeptide, are important tools for the engineering of proteins for diagnostic and therapeutic applications. We have recently described a selection strategy called covalent DNA display, in which individual proteins are covalently linked to the cognate encoding DNA template in separate droplets of a water-in-oil emulsion. We here report on the optimization of several experimental steps in covalent DNA display technology, such as the elution conditions and the PCR strategy used for the amplification of selected DNA templates. A PCR assembly strategy was developed, which allows the amplification of the DNA templates over repeated rounds of selection. In addition, we could demonstrate that approximately 50% of the DNA templates form a covalent adduct with the corresponding proteins in the compartments of a water-in-oil emulsion. In model selection experiments, differences in recovery efficiency <100 000 per round of selection could be observed when comparing a specific binding polypeptide with a binder of irrelevant specificity. Furthermore, the optimized protocol was successfully applied for the selection of single domain proteins, capable of specific binding to mouse serum albumin (MSA). A mutant derived from the SH3 domain of the Fyn kinase, with millimolar affinity to MSA, was affinity matured using covalent DNA display and yielded several MSA binding FynSH3 variants with dissociation constants in the 100 nM range.     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.     

Rapid isolation of high-affinity protein binding peptides using bacterial display

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.     

Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis

One approach to the analysis of leucocyte cell surface proteinsis to express their domains with part of another protein asa carrier. We report the use of two immunoglobulin superfamily(IgSF) domains from rat CD4 (CD4d3+4) in producing domains fromvarious superfamilies as chimeric proteins in Chinese hamsterovary cell lines. Four types of construct were successfullyexpressed containing: (i) the two IgSF domains of CD48; (ii)the IgSF domain of mb-1 which is part of the B cell antigenrecognition complex; (iii) a T cell receptor V domain; and (iv)the N-terminal domain of CD5 which belongs to the scavengerreceptor superfamily. This CD5 chimeric protein was antigenkfor a panel of CD5 mAbs showing that mAbs with functional effectsreacted with the N-terminal domain of CD5. The CD48 chimericprotein has been used both as multivalent complexes producedby crosslinking with mAbs recognizing CD4 and in a monomericform to analyse the kinetics of the interaction between CD48and CD2 [van der Merwe et al. (1993) EMBO J., 12, 4945–4954].     

Engineering aggregation-resistant proteins by directed evolution

A method was recently described for selecting aggregation-resistant antibody domains. A repertoire of domains displayed on filamentous bacteriophage were heated/cooled and selected for binding to the affinity ligand protein A specific for the folded domains. Here we describe a generalization of the method based on the selection for retained phage infectivity and for the binding of an appended sequence tag, and applicable to any protein displayable in multivalent form on phage.     

Negatively charged purification tags for selective anion-exchange recovery

A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.     

In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.     

A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry

Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host ( approximately 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.     

Large-scale modelling as a route to multiple surface comparisons of the CCP module family

Numerous mammalian proteins are constructed from a limited repertoire of module-types. Proteins belonging to the regulators of complement activation family--crucial for ensuring a complement-mediated immune response is targeted against infectious agents--are composed solely of complement control protein (CCP) modules. In the current study, CCP module sequences were grouped to allow selection of the most appropriate experimentally determined structures to serve as templates in an automated large-scale structure modelling procedure. The resulting 135 individual CCP module models, valuable in their own right, are available at the online database http://www.bru.ed.ac.uk/~dinesh/ccp-db.html. Comparisons of surface properties within a particular family of modules should be more informative than sequence alignments alone. A comparison of surface electrostatic features was undertaken for the first 28 CCP modules of complement receptor type 1 (CR1). Assignments to clusters based on surface properties differ from assignments to clusters based on sequences. This observation might reflect adaptive evolution of surface-exposed residues involved in protein-protein interactions. This illustrative example of a multiple surface-comparison was indeed able to pinpoint functional sites in CR1.     

A combinatorial library of an {alpha}-helical bacterial receptor domain

The construction and characterization of a combinatorial libraryof a solvent-exposed surface of an -helical domain derived froma bacterial receptor is described. Using a novel solid-phaseapproach, the library was assembled in a directed and successivemanner utilizing single-stranded oligonucleotides containingmultiple random substitutions for the variegated segments ofthe gene fragment The simultaneous substitution of 13 residuesto all 20 possible amino acids was carried out in a region spanning81 nucleotides. The randomization was made in codons for aminoacids that were modelled to be solvent accessible at a surfacemade up from two of the three a-helices of a monovalent Fc-bindingdomain of staphylococcal protein A. After cloning of the PCR-amplifiedlibrary into a phagemid vector adapted for phage display ofthe mutants, DNA sequencing analysis suggested a random distributionof codons in the mutagenized positions. Four members of thelibrary with multiple substitutions were produced in Escherichiacoli as fusions to an albumin-binding affinity tag derived fromstreptococcal protein G. The fusion proteins were purified byhuman serum albumin affinity chromatography and subsequentlycharacterized by SDSelectrophoresis, CD spectroscopy and biosensoranalysis. The analyses showed that the mutant protein A derivativescould all be secreted as soluble full-length proteins. Furthermore,the CD analysis showed that all mutants, except one with a prolineintroduced into helix 2, have secondary structures in closeagreement with the wild-type domain. These results proved thatmembers of this -helical receptor library with multiple substitutionsin the solvent-exposed surface remain stable and soluble inE.coli. The possibility of using this library for a phenotypicselection strategy to obtain artificial antibodies with novelfunctions is discussed.     

Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.