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PubMed1999

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization.

We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10(-8 )M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.

Amino Acid Sequence↗
PubMed1991

Electrophysiological activity of the C-peptide of the Locusta insulin-related peptide. Effect on the membrane conductance of Locusta neurones in vitro.

The C-peptide of Locusta insulin-related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5-kDa peptide, has been synthesised chemically by the solid-phase method, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1-38 residue-related fragment, named 4-kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5-kDa and 4-kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage-clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5-kDa)1, activated at potentials more hyperpolarised than -50 mV, peaked at about -75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5-kDa)2 was activated at potentials more depolarised than -50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5-kDa and 4-kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.

Animals↗
PubMed1995

Putative mechanism for guinea pig ileum contraction by N-formyl peptides. A comparative study of N-formyl and N-acetyl peptides with the N-terminal sequence of the calpain small subunit.

N-formyl and N-acetyl peptides with the N-terminal sequence of the calpain small subunit were prepared and their spasmogenic activity was examined using guinea pig ileum preparations. Sections of ileum were found to contract in the presence of all N-formyl peptides used (tri- to nonapeptides and tridecapeptide) but failed to contract with N-acetyl peptides, although both N-formyl and N-acetyl peptides have chemotactic activity, indicating that spasmogenic activity and chemotactic activity involve different mechanisms. A formyl peptide antagonist, Boc-Phe-Leu-Phe-Leu-Phe, suppressed contraction by formyl peptides whereas a histamine antagonist, diphenhydramine, suppressed contraction by formyl peptides as well as by histamine. In addition, formyl peptide-induced contractions were noted after an approximately 20-sec time lag, and their profiles were bell-shaped and roughly symmetrical. On the other hand, histamine- and acetylcholine-induced contractions exhibited a much shorter time lag. These data led us to conclude that contraction induced by formyl peptides may not occur as a direct response but may be due to the histamine released from mast cells present in the tissues of the small intestine.

Amino Acid Sequence↗
PubMed1992

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Amino Acid Sequence↗
PubMed1990

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

Amino Acid Sequence↗
PubMedDate not supplied

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment.

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.

Animals↗
PubMed1995

Detailed motifs for peptide binding to HLA-A*0201 derived from large random sets of peptides using a cellular binding assay.

Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A*0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A*0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at positions 4 for binding, and the deleterious effect of R at position 5 (in 9-mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A*0201 by computer analysis (see accompanying paper [56]).

Alleles↗
PubMed1996

The impact of DR3 microvariation on peptide binding: the combinations of specific DR beta residues critical to binding differ for different peptides.

HLA-DR molecules are a group of highly polymorphic glycoprotein heterodimers that present peptide antigens to T lymphocytes for immune surveillance. To assess the significance of limited polymorphism on the functional differentiation of DR molecules, the binding of several immunogenic peptides to the DR3 microvariants [DR(alpha, beta 1*0302) and DR(alpha, beta 1*0301)] and to mutants of these DR3 molecules was examined. This analysis has shown that each residue (DR beta 26, DR beta 28, DR beta 47, and DR beta 86), which differentiates these two DR3 molecules, contributes to their functional distinction and that the relative contribution of each residue varies for different peptide/DR3 complexes. For example, DR beta 28 and DR beta 86 controlled the mycobacterium tuberculosis 65-kD heat shock protein peptides 3-13 and 4-15 (HSP) binding specificity to DR (alpha, beta 1*0301). [HSP does not bind to DR(alpha, beta 1*0302)], whereas DR beta 26, DR beta 28, and DR beta 86 controlled the influenza hemagglutinin peptide 306-318 (HA) binding specificity to DR(alpha, beta 1*0302). [HA does not bind to DR(alpha, beta 1*0301).] In comparison, DR beta 86 alone controlled the binding level difference of sperm whale myoglobin peptide 132-151 (SWM) and of myelin basic protein peptide 152-170 (MBP) [both bind to DR(alpha, beta 1*0301) at levels five times greater than to DR(alpha, beta 1*0302)] to the DR3 molecules. Although not critical, additional DR beta residues influenced the binding level of individual peptides of each of the DR3 molecules and, again, the combinations of these residues differed for different peptide/DR3 complexes. These data showed that individual DR residues vary in their relative contribution to the interaction between a specific DR molecule and different peptides and that limited polymorphism can create substantial differences in the peptide binding profiles among DR molecules.

Amino Acid Sequence↗
PubMed1990

Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y-immunoreactive fibres are present in normal synovium but depleted in patients with rheumatoid arthritis.

By means of antisera to cytoplasmic components of nerve fibres and neuropeptides which are known to be present in sensory or sympathetic nerves we have examined the distribution of both total and different types of nerve fibres in normal and inflamed human synovial tissue. Samples of synovia were obtained at surgery from five normal and five rheumatoid patients (age range 10-77 years). In order to map the overall neural innervation of the synovium, antiserum to the general neuronal marker protein gene product 9.5 was employed. Substance P and calcitonin gene-related peptide antisera were employed to identify sensory fibres and antisera to the C-flanking peptide of neuropeptide Y to distinguish sympathetic nerves. In normal synovium protein gene product 9.5-immunoreactive fibres were numerous, in particular, the vasculature was densely innervated. Free protein gene product 9.5-immunoreactive fibres were less numerous but were present in all synovia examined, and in many cases these extended to the intimal layer. Neuropeptide immunostaining was predominantly found in perivascular networks. Fibres immunoreactive for the C-flanking peptide of neuropeptide Y were exclusively located around blood vessels whereas free fibres were immunoreactive for substance P or calcitonin gene-related peptide. As with free protein gene product 9.5-immunoreactive fibres, fibres expressing substance P or calcitonin gene-related peptide immunoreactivity were often seen in the intimal cell layer. In rheumatoid arthritis a similar innervation to that seen in normal synovium was apparent in the deep tissue but fibres immunoreactive for protein gene product 9.5, the C-flanking peptide of neuropeptide Y, substance P or calcitonin gene-related peptide were not visible in the more superficial tissues or the intimal cell layer. In addition, immunostaining of neuropeptides in the deep tissue was weaker in the diseased tissues than in normal controls. The data unequivocally demonstrate that synovial tissues are richly innervated and confirm the presence of both sensory and sympathetic nerves. The absence of nerves which innervate the superficial synovium in rheumatoid arthritis might suggest that there is increased release of substance P, calcitonin gene-related peptide and the C-flanking peptide of neuropeptide Y, reducing the stores in the nerves to levels below that detectable by immunocytochemistry. However, since protein gene product 9.5-immunoreactive nerves were not seen in the inflamed tissue it is probable that synovial growth outflanks neural growth and consequently as the disease progresses neural structures become restricted to deeper tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2005

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

Amino Acid Substitution↗
PubMed2006

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location.

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid side-chains at the N- and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N- and C-termini of a model peptide sequence, where the functional end-groups were N(alpha)-acetyl-X- and N(alpha)-amino-X- at the N-terminus and -X-C(alpha)-carboxyl and -X-C(alpha)-amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (between the -X-C(alpha)-carboxyl and -X-C(alpha)-amide peptide series) for hydrophobic side-chains. A similar result was seen for the N(alpha)-acetyl-X- and N(alpha)-amino-X- peptide series, where the largest differences in coefficient values were observed for hydrophobic side-chains. Coefficients derived from substitutions at the C-terminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and coefficients for the C-terminal residues. For prediction of peptide retention time, the sum of the coefficients must include internal and terminal coefficients.

Amino Acid Sequence↗
PubMed2005

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides.

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.

Electron Spin Resonance Spectroscopy↗
PubMed2001

Plasticity in protein-peptide recognition: crystal structures of two different peptides bound to concanavalin A.

The structures of concanavalin A (ConA) in complex with two carbohydrate-mimicking peptides, 10-mer (MYWYPYASGS) and 15-mer (RVWYPYGSYLTASGS) have been determined at 2.75 A resolution. In both crystal structures four independent peptide molecules bind to each of the crystallographically independent subunits of ConA tetramer. The peptides exhibit small but significant variability in conformations and interactions while binding to ConA. The crystal structure of another similar peptide, 12-mer (DVFYPYPYASGS), in complex with ConA has been determined (Jain, D., K. J. Kaur, B. Sundaravadivel, and D. M. Salunke. 2000. Structural and functional consequences of peptide-carbohydrate mimicry. J. Biol. Chem. 275:16098-16102). Comparison of the three complexes shows that the peptides bind to ConA at a common binding site, using different contacting residues and interactions depending on their sequence and the local environment at the binding site. The binding is also optimized by corresponding plasticity of the peptide binding site on ConA. The diversity in conformation and interactions observed here are in agreement with the structural leeway concerning plasticity of specific molecular recognition in biological processes. The adaptability of peptide-ConA interactions may also be correlated with the carbohydrate-mimicking property of these peptides.

Concanavalin A↗
PubMed1999

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.

Amino Acid Sequence↗
PubMed2001

Synthetic peptides as inactivators of multimeric enzymes: inhibition of Plasmodium falciparum triosephosphate isomerase by interface peptides.

Synthetic peptides corresponding to two distinct segments of the subunit interface of the homodimeric enzyme triosephosphate isomerase (residues 9-18, ANWKCNGTLE, peptide I; residues 68-79, KFGNGSYTGEVS, peptide II) from Plasmodium falciparum (PfTIM) have been investigated for their ability to act as inhibitors by interfering with the quaternary structure of the enzyme. An analog of peptide II containing cysteine at the site corresponding to position 74 and tyrosine at position 69 in the protein sequence KYGNGSCTGEVS (peptide III) was also investigated. A substantial fall in enzyme activity was observed following incubation of the enzyme with peptide II, whereas peptide I did not show any appreciable inhibition. The inhibitory effect was more pronounced on two mutants of PfTIM (Y74C and Y74G), both of which have reduced stability compared to the wild-type protein due to an interface cavity. The IC50 value determined for peptide II is in the range of 0.6-0.8 microM. This study suggests that interface peptides of oligomeric enzymes can be used to inhibit dimeric enzymes by disrupting their native multimeric states and may provide lead structures for potential inhibitor design.

Amino Acid Sequence↗
PubMed1997

Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity.

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

Amino Acid Sequence↗
PubMed1988

Effect of peptide chain length on peptide retention behaviour in reversed-phase chromatography.

The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time and/or the elution order on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption generally agrees well for small peptides (up to ca. 15 residues), the retention times of increasingly larger peptides are less than expected from a simple summation of retention coefficients. In the present study, we report the synthesis of four series of peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues, Ac = acetyl): Ac-(G-L-G-A-K-G-A-G-V-G)n-amide (n = 1-5), Ac-(G-K-G-L-G)n-amide (n = 1, 2, 4, 6, 8, 10), Ac-(L-G-L-K-A)n-amide (n = 1, 2, 4, 6, 8, 10) and Ac-(L-G-L-K-L)n-amide (n = 1, 2, 4). From the retention behaviour of these peptide polymers on C4, C8 and C18 stationary phases under gradient elution conditions, we have clearly established the effect of polypeptide chain length and hydrophobicity on peptide retention. This, in turn, has enabled us to extend the utility of retention time prediction for peptides containing up to 50 residues by introducing a peptide chain-length correction.

Chromatography, High Pressure Liquid↗
PubMed1997

A novel, highly efficient peptide-HLA class I binding assay using unfolded heavy chain molecules: identification of HIV-1 derived peptides that bind to HLA-A*0201 and HLA-A*0301.

A novel cell-free, highly automated peptide-HLA binding assay has been designed during which a mixture of unfolded recombinant HLA heavy chain molecules, beta 2-microglobulin and a fluorescent labeled standard peptide is allowed to form peptide-HLA complexes. The binding of a peptide of interest is monitored as the ability to inhibit the formation of fluorescent peptide-HLA complexes. The assay was validated using published, known HLA-A* 0201 and HLA-A* 0301 binding peptides. In addition a selected set of HIV-1LAI reverse transcriptase derived 10-mer peptides, that had been selected on the basis of HLA-A* 0201 or HLA-A* 0301 binding motifs, were tested for HLA-A* 0201/A* 0301 binding. In that set we identified 8 peptides which bound with high affinity to HLA-A* 0201 and 5 peptides which bound with high affinity to HLA-A* 0301. The major advantage of the use of denatured heavy chain is the improved economy and efficiency, as unfolded protein material is in principle easily accessible by recombinant technology.

HIV Reverse Transcriptase↗