The integration of ProxiMAX randomisation with CIS display for the production of novel peptides

Saturation mutagenesis is a powerful tool in modern protein engineering, which permits key residues within a protein to be targeted in order to potentially enhance specific functionalities. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK/S) has inherent redundancy and consequent disparities in codon representation. Therefore, both chemical (trinucleotide phosphoramidites) and biological methods (sequential, enzymatic single codon additions) of non-degenerate saturation mutagenesis have been developed in order to combat these issues and so improve library quality. Large libraries with multiple saturated positions can be limited by the method used to screen them. Although the traditional screening method of choice, cell-dependent methods, such as phage display, are limited by the need for transformation. A number of cell-free screening methods, such as CIS display, which link the screened phenotype with the encoded genotype, have the capability of screening libraries with up to 1014 members. This thesis describes the further development of ProxiMAX technology to reduce library codon bias and its integration with CIS display to screen the resulting library. Synthetic MAX oligonucleotides are ligated to an acceptor base sequence, amplified, and digested, subsequently adding a randomised codon to the acceptor, which forms an iterative cycle using the digested product of the previous cycle as the base sequence for the next. Initial use of ProxiMAX highlighted areas of the process where changes could be implemented in order to improve the codon representation in the final library. The refined process was used to construct a monomeric anti-NGF peptide library, based on two proprietary dimeric peptides (Isogenica) that bind NGF. The resulting library showed greatly improved codon representation that equated to a theoretical diversity of ~69%. The library was subsequently screened using CIS display and the discovered peptides assessed for NGF-TrkA inhibition by ELISA. Despite binding to TrkA, these peptides showed lower levels of inhibition of the NGF-TrkA interaction than the parental dimeric peptides, highlighting the importance of dimerization for inhibition of NGF-TrkA binding.

Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling

BACKGROUND/AIMS: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. METHODS: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. RESULTS: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. CONCLUSION: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

Self-condensation of a thiazole-peptide bearing a 21-membered loop into a library of giant macrocycles with multiple orthogonal loops

Tetrapeptide analogue H-[Glu-Ser-Lys(Thz)]-OH, containing a turn-inducing thiazole constraint, was used as a template to produce a 21-membered structurally characterized loop by linking Glu and Lys side chains with a Val-Ile dipeptide. This template was oligomerized in one pot to a library (cyclo-[1](n), n = 2-10) of giant symmetrical macrocycles (up to 120-membered rings), fused to 2-10 appended loops that were carried intact through multiple oligomerization (chain extension) and cyclization (chain terminating) reactions of the template. A three-dimensional solution structure for cyclo-[1](3) shows all three appended loops projecting from the same face of the macrocycle. This is a promising approach to separating pepticle motifs over large distances.

A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers : a potential marker of physical activity

Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic / anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3 / endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk / survival.

Mechanism‐based selection of a potent kallikrein‐related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI‐1

Potent and specific enzyme inhibition is a key goal in the development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclized peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein-related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through the use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry. This approach identified a single dominant inhibitor species with molecular weight of 1562.4 Da, which is consistent with the SFTI variant SFTI-WCTF. Once synthesized individually this inhibitor showed an IC50 of 173.9 ± 7.6 nM against chromogenic substrates and could block protein proteolysis. Molecular modeling analysis suggested that selection of SFTI-WCTF was driven by specific aromatic interactions and stabilized by an enhanced internal hydrogen bonding network. This approach provides a robust and rapid route to inhibitor selection and design.

Interaction of A beta peptide (1-40) with amino acid aluminium complexes: relevance to Alzheimer's disease

A beta (39-43 aminoacid residues) is the principal peptide component of amyloid deposits in Alzheimer's disease (AD). A beta peptide is derived from the amyloid precursor protein (APP) in which mutations give rise to many forms of familial AD. Aluminium is reported to play a key role in inducing conformational change in the synthetic beta-amyloid peptide (1-40)from alpha-helix to beta-pleated sheet, leading to aggregation and fibrillar formation. We have studied the interaction of amino acid-Al complexes such as D-Asp-Al and L-Glu-Al with A beta(1-40) in TFE/buffer (70% TFE and 30% H2O v/v pH 6.7) mixture using CD spectroscopy. The interaction of either of these amino acid complexes with A beta(1-40) results in loss of alpha-helical content and the peptide is more unstructured compared to free Al3+ in the solution. Our data strongly support the idea, that the Al3+ in the form of aminoacid-Al complexes is more effective in inducing random coil conformation in the A beta peptide than the free Al3+ present in the solution.

Effect of signal peptide on stability and folding of escherichia coli thioredoxin

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

Recognition of Membrane-Bound Fusion-Peptide/MPER Complexes by the HIV-1 Neutralizing 2F5 Antibody : Implications for Anti-2F5 Immunogenicity

13 p.

Developing peptide based capture agents for diagnostics and therapeutics

Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.