Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Summary Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent,N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphateN-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

Human erythrocyte contains a factor that stimulates the peptidase activities of multicatalytic proteinase complex.

A novel biological factor that stimulates the peptidase activities of multicatalytic proteinase complex (MPC) has been identified and partially purified from human erythrocytes. The stimulatory factor enhances trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing activity of MPC in a dose related manner. At saturating concentration of the stimulatory factor, MPC increases the activity to a different extent (10 to 56 fold) depending on the substrate used to assay the enzyme. The stimulatory factor does not hydrolyze neither amino-blocked peptides which are used to assay MPC nor typical substrates for amino and diamino-peptidases. The stimulatory factor is characterized by a high molecular mass (300 kDa) and an extreme instability since it loses the activity at 46 degrees C in 10 min and at 4 degrees C within a week. The stimulatory activity is inactivated by incubation in acidic or alkaline media, and by treatment with protease V8, but it is relatively resistant to the action of trypsin. It has been suggested that the novel stimulatory factor herein described is a protein or a protein complex which may modulate the function and the activity of MPC by association-dissociation interaction.     

Haloacetyl groups as reversible protection of the amino function: cleavage with 2-aminothiophenol.

Haloacetylamino acids and haloacetyl peptides react rapidly with 2-aminothiophenol in weakly alkaline media to yield 2-aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N-haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloroacetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates. Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2-haloacids to modern polypeptide chemistry are outlined.     

A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain

Peptide antigens used to generate site-specific antibodies to proteins are of interest in the development of vaccines. The need to conjugate them to a carrier protein for optimal immunogenicity results in a number of problems including a possible immune response to the carrier. Here we describe a new method of synthesizing an immunogenic peptide antigen, referred to as multiple antigenic peptide (MAP), which may render the need for a carrier protein obsolete. A 14-residue sequence derived from the human T cell antigen receptor beta-chain constant region was selected, and the peptide was synthesized directly onto a branching lysine core with 8 copies of the 14-residue peptide linked to the core by the COOH-terminal amino acid. The molecular weight of this structure was 13,422 of which only 7% represents the lysine residues of the core. The octameric MAP was highly immunogenic in mice and rabbits, allowing production of polyclonal and monoclonal antibodies. The majority of these antibodies reacted with the peptide in its monomeric form as well as its octameric form. Moreover, the antibodies reacted with the intact beta-chain protein. The antigenic determinants of the peptide that were recognized by the antibodies included continuous determinants and conformational determinants. The NH2-terminal residues of the octameric MAP appeared to be most immunogenic. There were no antibodies to the central lysine core. This method of direct synthesis of a polymeric peptide provides accurate knowledge of the conformation and quantity of the peptide prior to immunization, which is usually not the case when peptides are conjugated to carriers. The method is versatile because the possibility exists to synthesize MAP with 16 or 32 peptide arms or to synthesize polymers containing two different peptides.     

Synthesis of a New Template with a Built-in Adjuvant and Its Use in Constructing Peptide Vaccine Candidates Through Polyoxime Chemistry

Synthetic lipopeptides are showing promise as vaccine candidates, but until now it has been very difficult to prepare them in homogeneous form. We describe the synthesis and characterization of a new water-soluble, four-branched template with a built-in lipophilic adjuvant (Pam₃Cys). Through the use of oxime chemistry, we attached four copies of an unprotected influenza virus peptide and characterized the product (13kDa) by reversed-phase HPLC and electrospray ionization mass spectrometry. Several other such constructions were made using the new template and different peptides. We seem to have a general method for making synthetic lipopeptides in homogeneous form.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.     

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.     

A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics

Current anticoagulants target coagulation factors upstream from fibrin assembly and polymerization (i.e., formation of fibrin clot). While effective, this approach requires constant patient monitoring since pharmacokinetics and pharmacodynamics vary from patient to patient. To address these limitations, we developed an alternative anticoagulant that effectively inhibits fibrin polymerization. Specifically, we investigated PEGylated fibrin knob “A” peptides, evaluating the effect of both polyethylene glycol (PEG) chain length (0, 2, 5, and 10–30 kDa) and knob peptide sequence (GPRPAAC, GPRPFPAC, and GPRPPERC) on inhibiting fibrin polymerization (i.e., clot formation). Thrombin‐initiated clotting assays with purified fibrinogen were performed to compare clot formation with each peptide–PEG conjugate. Results indicated a biphasic effect of PEG chain length, whereby, active‐PEG conjugates demonstrated increasingly enhanced inhibition of fibrin polymerization from 0 to 5 kDa PEG. However, the anticoagulant activity diminished to control levels for PEG chains above 5 kDa. Ultimately, we observed a 10‐fold enhancement of anticoagulant activity with active peptides PEGylated with 5 kDa PEG compared to non‐PEGylated knob peptides. The sequence of the active peptide significantly influenced the anticoagulant properties only at the highest 1:100 molar ratio where GPRPFPAC‐5 kDa PEG and GPRPPERC‐5 kDa PEG demonstrated significantly lower percent clottable protein than GPRPAAC‐5 kDa PEG. Moreover, human plasma treated with the active 5 kDa PEG conjugate exhibited delayed prothrombin time to within the therapeutic range specified for oral anticoagulants. Collectively, this study demonstrated the utility of PEGylated fibrin knob peptides as potential anticoagulant therapeutics. Biotechnol. Bioeng. 2011;108: 2424–2433. © 2011 Wiley Periodicals, Inc.     

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98kDa as a monomer and 13.96kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.     

Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2-microglobulin translated in vitro

We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2-microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly.     

Synthetic peptide epitope-based polymers: controlling size and determining the efficiency of epitope incorporation.

The assembly of synthetic peptide-based vaccines that incorporate multiple epitopes is a major goal of vaccine development, because such vaccines will potentially allow the immunization of outbred populations against a number of different pathogens. We have shown that free radical-induced polymerization of individual peptide epitopes results in the incorporation of multiple copies of the same or different epitopes into high molecular weight immunogens (O'Brien-Simpson, N.M., Ede, N.J., Brown, L.E., Swan, J. & Jackson, D.C. (1997) Polymerization of unprotected synthetic peptides: a view toward synthetic peptide vaccines. J. Am. Chem. Soc.119, 1183-1188; Jackson, D.C., O'Brien-Simpson, N., Ede, N.J. & Brown, L.E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15, 1697-1705). The ability to control the size of these polymers, to determine the physical and chemical properties of the backbone material and also to know the extent to which individual peptide epitopes are incorporated are important manufacturing considerations and form the subject of this study. We show here that the polymerization process is highly efficient with at least 70% of peptides incorporated into the resulting polymer, that acrylamide and acryloylated amino acids can be used as comonomers with peptide epitopes in the polymerization reaction and that the choice of the comonomer can influence the properties of the resulting polymer. We also show that the size of chain growth polymers is restricted in the presence of chain transfer agents, that the resulting polymer size can be predicted and that there is little or no difference in the immunogenicity of polymers that range in apparent molecular size between 18 kDa and 335 kDa. The successful polymerization of peptide epitopes with an acryloyl-amino acid creates the potential for introducing different physical and chemical properties into artificial protein immunogens.