Biomedical subjects

G J Cotton

Publications and source records attributed to G J Cotton.

8 recordsLinked to original sources

PubMed2001

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity.

Fluorescent biosensors hold great promise for drug discovery. Using a solid-phase version of protein semi-synthesis, we incorporated two fluorophores at specific sites within a truncated version of the c-Crk-II protein. The resulting fluorescent protein biosensor permits the real-time monitoring of Abl kinase activity and provides a robust and rapid method for assaying Abl kinase inhibitors.

Amino Acid Sequence↗

PubMed2000

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation.

BACKGROUND: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation. RESULTS: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process. CONCLUSIONS: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.

Animals↗

PubMed1999

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Amino Acid Sequence↗

PubMed1999

Peptide ligation and its application to protein engineering.

The ability to assemble a target protein from a series of peptide fragments, either synthetic or biosynthetic in origin, enables the covalent structure of a protein to be modified in an unprecedented fashion. The present technologies available for performing such peptide ligations are discussed, with an emphasis on how these methodologies have been utilized in protein engineering to investigate biological processes.

Animals↗

PubMed1999

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.

Amino Acid Sequence↗

PubMed1998

Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cell apoptosis and block cell death induced by gp120.

A vaccine against HIV-1 virus would block initial infection and must target conserved residues. Since initial infection depends on binding of the viral envelope protein gp120 to CD4 on the cell surface, the CD4 binding site of gp120 is a target for vaccine design. To identify the optimal biologically active site, we synthesized a series of 32-mer peptides, based on conserved residues in the C3 and C4 regions of gp120. These included three of five sequence discontinuous residues known to be involved in CD4 binding, one or two of which were substituted with alanine. We also synthesized a 44-mer peptide with an additional branch to incorporate an extra C4 region sequence including a fourth CD4 binding residue. All these peptides used an oxidized Cys-X-Cys bridge to link the discontinuous sequence elements in a manner suggested by the known conserved disulfide bridges in gp120. Polyclonal sera raised to these peptides indicate that they all contain both B and T lymphocyte epitopes. Binding of the peptides to CD4-transfected HeLa cells reveals a hierarchy dependent on the number of relevant CD4 binding residues present. Furthermore, antibody cross-linking of peptides bound to the surface of human T cells results in apoptosis that is similar to the known properties of gp120. The peptide incorporating three CD4 binding residues competitively inhibited gp 120-induced T lymphocyte apoptosis. Thus, we have synthesized novel, branched peptides incorporating conserved discontinuous sequences from two different conserved domains of HIV-1 gp120 that contain T and B lymphocyte epitopes and mimic biological functions of the native protein. These synthetic peptides are candidates for future vaccine development.

Amino Acid Sequence↗

PubMed1998

Chemical ligation of unprotected peptides directly from a solid support.

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid-phase ligation approach eliminates the need to manipulate peptide (alpha)thioacid and peptide (alpha)thioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an (alpha)thioester linkage. This can be achieved efficiently using optimized Boc-solid-phase peptide synthesis on a 3-mercaptopropionamide-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer support (HS-PEGA). Once the synthesis is complete, the fully protected peptide (alpha)thioester resin is treated with HF to give the corresponding fully unprotected peptide (alpha)thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide-resins were then used to perform both intramolecular (head-to-tail cyclizations) and intermolecular solid-phase ligations. The intramolecular solid-phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.

Amino Acid Sequence↗

PubMed1996

Design and synthesis of a highly immunogenic, discontinuous epitope of HIV-1 gp120 which binds to CD4+ve transfected cells.

Here we report the design and synthesis of a novel 32-mer peptide, Lys364-378Val445-459.oxidized (named GC-1), which represents a discontinuous epitope from the C3 and C4 domains of gp120 from the HIV-1 IIIB isolate. This peptide induces high titre IgG antibody responses in mice, indicating that it has both B and T cell epitopes. Epitope mapping using reduced GC-1 and appropriate linear peptides demonstrated that a large proportion of the antibodies raised in mice were directed against discontinuous epitope(s). Furthermore, antibodies to GC-1 peptide cross-reacted with purified HIV-1 strain IIIB gp120, indicating the GC-1 mimicked at least one epitope of the native protein. The peptide, which incorporates three gp120 residues Asp 368, Glu 370 and Asp 457, previously shown to be critical for CD4 ligation, bound to the surface of a CD4 transfected human epithelial cell line HeLa, but not to the parent cell line and inhibited binding of recombinant HIV-1 gp120 to recombinant soluble CD4. We have synthesized the first of a series of discontinuous peptides which will be useful for the probing of interactions of HIV-1 gp120 with the CD4 molecule.

Amino Acid Sequence↗