PNA-encoded protease substrate microarrays

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.     

Fluorous-based peptide microarrays for protease screening

As ever more protease sequences are uncovered through genome sequencing projects, efficient parallel methods to discover the potential substrates of these proteases becomes crucial. Herein we describe the first use of fluorous-based microarrays to probe peptide sequences and begin to define the scope and limitations of fluorous microarray technologies for the screening of proteases. Comparison of a series of serine proteases showed that their ability to cleave peptide substrates in solution was maintained upon immobilization of these substrates onto fluorous-coated glass slides. The fluorous surface did not serve to significantly inactivate the enzymes. However, addition of hydrophilic components to the peptide sequences could induce lower rates of substrate cleavage with enzymes such as chymotrypsin with affinities to hydrophobic moieties. This work represents the first step to creating robust protease screening platforms using noncovalent microarray interface that can easily incorporate a range of compounds on the same slide.     

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.     

Physicochemical properties and substrate specificity of protease C from dormant cotton seeds

The substrate specificity of a proteolytic enzyme — protease C — isolated from cotton seeds has been studied. The activity of protease C is suppressed completely under the action of diisopropyl phosphorofluoridate. Protein inhibitors — duck ovomucoid, soybean inhibitor, and also TPCK — suppressed the activity of protease C to different degrees. On the basis of results obtained in the hydrolysis of the cottonseed reserve proteins, 7S and 11S globulins, and the B chain of insulin, protease C has been assigned to the serine group of endopeptidases. The optimum conditions — pH, time, and temperature — at which protease C exhibits its maximum activity has been determined.Institute of Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 744–746, September–October, 1988.     

A combined SPS-LCD sensor for screening protease specificity

A hydrogel-based sensor for screening protease specificity has been developed that combines the versatility of solid-phase synthesis (SPS) with the simplicity of liquid crystal display (LCD) technology.     

Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

A method for rapid protease substrate evaluation and optimization

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.     

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K_D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

We introduce highly affine epitope-imprinted polymer-based microarrays for selective protein detection by surface plasmon resonance imaging as shown through the selective recognition of the receptor binding domain of SARS-CoV-2 spike protein.     

Studies on the substrate specificity of Escherichia coli galactokinase

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]     

A modular assembly strategy for improving the substrate specificity of small catalytic peptides

In contrast to large proteins, small peptide catalysts typically display limited specificity for small molecule substrates. This is presumably a result of the limited opportunities small peptides have to fold in a manner that provides for the formation of an isolated reaction vessel that effectively binds and sequesters substrates from bulk solvent while at the same time catalyzing their transformation. For the preparation of small peptide catalysts that possess improved substrate specificity, we have developed a modular assembly strategy that involves appending phage display-derived substrate binding-domain modules to catalytically active peptide domains. We demonstrate the potential of this strategy with the construction of a small 35-amino acid residue aldolase peptide with improved substrate specificity. The advantages of this approach are that it reduces the demand on the functionalization of the catalytic site and it is modular, therefore making its adaptation to a variety of specificities rapid. The modular assembly strategy studied here may present advantages over exhaustive searches of large random-sequence peptide libraries for peptides with singular function.     

Model based soft-sensor for on-line determination of substrate

A software sensor for on-line determination of substrate was developed based on a model for fed-batch alcoholic fermentation process and on-line measured signals of ethanol, biomass, and feed flow. The ethanol and biomass signals were obtained using a colorimetric biosensor and an optical sensor developed in previous works that permitted determination of ethanol at a concentration of 0–40 g/L and biomass of 0–60 g/L. The volume in the fermentor could be continuously calculated using the total measured signal of the feed flow. The results obtained show that the model used is adequate for the proposed software sensor and determines continuously the substrate concentration with efficiency and security during the fermentation process.     

Chemoenzymatic synthesis of prodigiosin analogues--exploring the substrate specificity of PigC

Analogues of prodigiosin, a tripyrrolic pigment produced by Serratia species with potent immunosuppressive and anticancer activities, have been produced by feeding synthetic analogues of the normal precursor MBC to mutants of Serratia sp. ATCC 39006 or to engineered strains of Escherichia coli; in this way it has been shown that the prodigiosin synthesising enzyme, PigC, has a relaxed substrate-specificity.     

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G?+?C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.