Construction of a photoactivatable profluorescent enzyme via propinquity labeling

A strategy for the construction of a profluorescent caged enzyme is described. An active site-directed peptide-based affinity label was designed, synthesized, and employed to covalently label a nonactive site residue in the cAMP-dependent protein kinase. The modified kinase displays minimal catalytic activity and low fluorescence. Photolysis results in partial cleavage of the enzyme-bound affinity label, restoration of enzymatic activity (60-80%) and a strong fluorescent response (10-20 fold). The caged kinase displays analogous behavior in living cells, inducing a light-dependent loss of stress fibers that is characteristic of cAMP action. This strategy furnishes molecularly engineered enzymes that can be remotely controlled in time, space, and total activity.     

Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.     

Screening of phage‐displayed human liver cDNA library against doxorubicin with drug‐immobilized monolithic polyacrylamide cryogel

Monolithic polyacrylamide cryogel was prepared and utilized as a new matrix for drug immobilization to screen against phage‐displayed human liver cDNA library. The macropores and hydrophilic nature of the cryogel made it possible for phage particles to pass unhindered. Doxorubicin, an anticancer drug, was covalently bonded to the monolithic cryogel by the glutaraldehyde method, and after five rounds of affinity selection performed in an SPE cartridge, phage clones that displayed Homo sapiens methyl CpG binding protein 2 (MeCP₂) were selectively enriched. The interaction between doxorubicin and MeCP₂ displayed phages was further validated by studying the retention of doxorubicin on MeCP₂ phage‐coupled cryogel. These results demonstrate that drug‐coupled polyacrylamide cryogel might be a promising kind of matrix for screening target proteins against phage‐displayed library. Copyright © 2013 John Wiley & Sons, Ltd.     

Manipulation of carrier proteins in antibiotic biosynthesis

Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.     

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals—The Etiological Factor of Chondrocalcinosis

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg²⁺-dependent and also works against soluble species of pyrophosphate.     

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…     

Optimisation d’une sélection in vitro d’enzymes

Optimisation of an in vitro enzyme selection. Isolation of a catalyst for a given chemical reaction may be achieved by in vitro selection of enzymes from a protein library. Here, we investigate the polymerisation reaction on filamentous phage and the cross-linking of substrate on phage to optimise an in vitro selection for DNA polymerase activity. The efficiency of the optimised selection is measured by enrichment factors up to 3.8 × 10³, the highest described so far for an in vitro selection of proteins for catalysis. It should be useful for directed polymerase evolution towards novel catalytic activities. To cite this article: E. Orsi, J.-L. Jestin, C. R. Chimie 6 (2003).     

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.     

A Method for the Selection of Catalytic Activity Using Phage Display and Proximity Coupling

Phage display has been used extensively for the selection of proteins with binding sites for ligands. Here, as illustrated with the example of DNA polymerase, the use of phage display for selection according to catalytic activity is described. Active enzymes are selected by binding of the reaction product P (see the scheme) cross-linked in the proximity of the enzyme E that catalyzed the reaction with the substrate S.     

Substrate turnover and inhibitor binding as selection parameters in directed evolution of blood coagulation factor Xa

A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.     

Combinatorial libraries of biocatalysts: application and screening

Enzymes can perform intricate regioselective and/or enantioselective chemical transformations and can accelerate reaction rates by enormous factors all under mild conditions. However, enzymes almost always present problems for use on an industrial scale. Evolutionary design approaches can be applied to the generation of stable enzymes with improved or novel catalytic activities. Directed evolution can be considered as the biotechnological equivalent of combinatorial chemistry, where the expressed proteins are the combinatorial libraries of biocatalysts. This review will focus on the search of novel biocatalysts produced by genetic engineering with modified activity and stability in different environments, substrate specificity and enantioselectivity. Methods of screening and/or selection for the desired properties will also be described. Finally, the efforts in de novo enzyme design are mentioned.     

Accelerated screening of phage-display output with alkaline phosphatase fusions

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.     

Phage-chips for novel optically readable tissue engineering assays

We report novel phage-based array chips that are optically readable for cell proliferation and morphology assays. Using M13 phages that were engineered to display RGD on its major coat proteins and/or immobilize FGFb on its minor coat proteins, we prepared arrays of phage spot matrices composed of self-assembled nanofibrous network structures. We cultured fibroblasts on the arrays and, using surface plasmon resonance (SPR) spectroscopy, monitored the effects of the biochemical cues displayed by the phage on cell proliferation and morphology. This study demonstrates the utility of engineered phages as promising coating materials for lab-on-a-chip (LOC) platforms, allowing sensitive monitoring of the effects of functional peptides on cell growth. Phage-chips have great potential for use as high-throughput screening systems for biochemical assays and biosensors and the discovery of novel drugs.     

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.     

In vitro selection for catalytic activity with ribosome display

We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.     

Analyzing and monitoring of phage–bacteria interaction using CE

The utilization of CE for monitoring bacteria–phage interaction was investigated in this study. Streptococcus thermophilus and Lactobacillus bulgaricus strains and their phages were used as model bacteria and phages for the purpose of validation in this study. CE with heterogeneous polymer polyethylene oxide was utilized for the separation of intact bacteria and investigation of phage–bacteria interaction. An intact phage detection was carried out with CZE by adding SDS in the running buffer. Calibration graphs of bacteria and phages were obtained with R² values of 0.963 and 0.937, respectively. S. thermophilus strain was infected with its virulent phage B3‐X18 for investigation of phage–bacteria interaction. It was observed in capillary electropherogram that the culture was lysed depending on the multiplicity of infection value and it showed to be completely lysed when the multiplicity of infection value was 10. The interaction of S. thermophilus strain with L. bulgaricus phage was also investigated by using a CE and a microbiological method and it was observed that the L. bulgaricus phage attached itself to the cell wall of S. thermophilus strain without damaging the cell.