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trifluoromethanesulfonic acid (TFMSA), in presence of suitable scavengers, anisole [30,31]. The Boc scheme has proven to be successful for peptide synthesis for decades, but the use of dangerous chemicals, harsh final acidic cleavage conditions and specially designed work place with skilled handler, provided an impetus to researchers for the development of milder alternatives (Figure 1). Figure 1: Schematic diagram showing Synthesis of peptide using t-Boc and Fmoc chemistry. In Fmoc/tBu, the Fmoc (9- fluorenyl methoxycarbonyl) group is used for the protection of the N-α amino group and the tert-butyl group for the side chains of several amino acids [32]. In Fmoc strategy, N-terminal Fmoc group can be removed by a variety of secondary amines, usually 20-50% piperidine in N, N-dimethylformamide (DMF) and there is no neutralization needed after deprotection while tert-butyl groups which are used to protect side chains of Fmoc amino acids, are cleaved by trifluoroacetic acid which also cleaves the peptide from the resin. Moreover, use of DMF as a solvent improved the yield of peptide. The need of milder conditions and easy handling of chemical used in Fmoc strategy makes it more popular than t-Boc strategy [Figure 1]. Moreover, synthetic peptides also showed immunological response in animal models. But, being small in size and simple structure, they are not found to be good immunogen until incorporated with some carrier/adjuvants. Tam et al in 1988 developed a method of synthesizing multiple antigen peptides (MAP) in an oligomeric form and as a macromolecule by the solid-phase peptide synthesis method. The MAPs consist of multiple copies of peptides circumsporozoite proteins of two species of malaria that are synthesized as single units on a branching lysyl matrix using a solid-phase peptide found to be more immunogenic as compared to a monomeric peptide [33-35]. In 1990, Tam et al. synthesized MAP containing four copies of peptide antigen [B(4), T(4), BT(4) and TB(4) epitopic arrangement] separately on a tertbutoxycarbonyl (Boc)-Ala-Pam resin using three Boc-Lys(Boc) amino acid as a starting point. Similarly, they also synthesized MAP models containing eight copies of the antigens [T(8), B-(8), BT-(8), and TB-(8)], the core was synthesized with seven lysines prepared from branching with three levels of Boc-Lys(Boc). In the MAP models containing eight copies of B or T antigens and one copy of T or B antigen [B-(8)-T, T(8)-B], the B or T antigen was first synthesized linearly on the Boc-Ala-Pam resin and then branched with three levels of lysine to give subsequent eight copies of T or B antigens. All peptides and MAPs were cleaved from the resin supports by low/high HF procedure (Figure 2) [36]. In 1992, Jean-Paul Briand et al. developed several MAP systems containing eight copies of 6-15 residue-long peptides derived from selected regions of various proteins. They observed that immunogenicity of MAPs was found to be superior than the same peptides linked to carrier protein by means of conventional conjugation procedures [37]. Multiple antigen peptide (MAP) consisted of a malaria T-cell epitope or of a human universal tetanus toxin helper T-cell epitope collinearly synthesized with B-cell epitopes from the circumsporozoite proteins of different malaria parasites stimulated specific T-cell clones [38]. Also, MAP system, consisted of an oligomeric branching lysine core to which B and T cell epitopes of meningococcal class 1 protein were attached as octameric and tetrameric dendron like structure, was synthesized and confers some conformational stability and predominance of antibodies directed towards conformational epitopes [39]. In the year 2000, Robert A. Boykins et al. developed a MAP containing multiple epitopes of human malaria parasite and the Tat protein of HIV type-1 (HIV-1-Tat) using Fmoc strategy [40]. Tetra-branched MAP containing four copies of peptide of the OMICS Group eBooks 006
24 amino acid extracellular domain of matrix protein 2 (M2e-MAP) of H5N1 virus strain VN/1194, a remarkably conserved across influenza A subtypes, was synthesized on [Fmoc-Lys(Fmoc)] 2 -Lys-Cys(Acm)-βAla-Wang Resin using Fmoc chemistry and cleaved of the peptide from the resin by treatment with trifluoroacetic acid (TFA), DTT, water, and triisopropylsila (TIPS) in the ratio 88:5:5:2, respectively. Crude peptide was purified by reversed phase high-performance liquid chromatography (RP-HPLC) and characterized by amino acid analysis and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). M2e-MAP vaccine induced strong MAP specific antibody response in murine model [41]. Fujiki T et al. in 2010, designed octavalent MAP containing eight copies of epitope of human tumor necrosis factor-α (TNF-α) synthesized on seven residues containing lysine backbone and in vitro developed TNF-α specific monoclonal antibodies in human peripheral blood mononuclear cells (PBMCs) [42]. Figure 2: Schematic diagram showing Synthesis of Multiple Antigen Peptide (MAP) using chemistry. Two chimeric multiple antigenic peptides (CMAP5 and CMAP8) and 4 monomeric linear peptides (LP1M, LP1N, LP1O, LP2) derived from the transmembrane proteins (gp41) of HIV-1 and HIV-2 were developed on a lysine core. CMAP5 and CMAP8 seropositivity with HIV-1 and 2 thereby, showing highly sensitive and specific assays for the detection of infections caused by HIV-1, including group M, N, and O, and HIV-2 [43]. Recently, our laboratory developed a MAP containing three B, one T cell epitopes of F1-Ag of Yersinia pestis on Fmoc–Gly–HMP–Tantagel resin using Fmoc chemistry. Fmoc-Lys (ivDde)-OH was incorporated at the beginning of the synthesis for each branch to serve as the branching point. Lysine is a unique amino acid having two amino terminals and thus it can support two N-terminal coupling reactions, thereby providing two groups with minimum stearic hindrance. ivDde is a protecting group which is resistant to piperidine, which is used to deprotect Fmoc group during synthesis. First sequence was added at the N-terminal end of first lysine and after completion, blocked with t-Boc protected amino acid to prevent further chain elongation. Use of t-Boc amino acid, being resistant to piperidine reaction, at the end of each sequence made possible to add new sequence without extending existing one. After completion of one sequence, ivDde group on ε-amino group of first lysine was deprotected by using hydrazine and another lysine (ivDde) was added at the same point. Next sequence was added on a-amino group of second lysine and subsequently completed the desired sequence before final deprotection with TFA [Figure 2] [44]. Biotechnology Approach for Peptide Production Peptides are of growing interest not only as therapeutics but also as transporters of non-peptidic molecules such as small molecule active pharmaceutical ingredient (API), cytotoxic or imaging agents. Increasing demand of peptide as therapeutics and major challenges associated with it has not only led to abundant innovations in stabilization or formulation technologies but also in manufacturing techniques. Producing therapeutic peptides is a complex and time-consuming process and can take several years just to identify it, determine its gene sequence, and validate a biotechnology process to manufacture it. There are certain challenges especially associated with the large-scale synthesis of peptides such as, to meet the production demand, proper strategy of synthesis applicable on all scale, downstream processing and isolation, quality control, regulatory implications, and production economics, which are needed to be OMICS Group eBooks 007
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Tyrosine Nitrated Proteins: Biochem
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[58-61]. Protein sulfhydryls [62] a
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2. Souza JM, Daikhin E, Yudkoff M,
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Oligoclonality of the antibody resp
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The Recent Methodologies of Protein
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are due to altering protein activit
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Figure 6: At Domain Level, Protein
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Brownian Dynamics (BD) method Prote
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protein demonstrate the lowest ener
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Figure 12: Steps in Protein Prepara
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FlexServ The flexibility of protein
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21. van der Kamp MW, Shaw KE, Woods
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Advances in Protein Thermodynamics
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3. Fluorescence correlation spectro
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Figure 3: Molecular Chaperone (Top
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Figure 7: A Potherse is Consists of
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Figure 12: Residue Clusters with Mu
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Figure 16: Various Features at the
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Figure 21: Stability of protein str
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Advances in Protein Chemistry Chapt
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MMPs are believed to remodel the EC
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MMPs7 (Matrilysin, PUMP 1) A big ro
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MMP27 (MMP-22, C-MMP) mRNAs for MMP
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Signal transduction MMP production
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a | Active MMPs are generated throu
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Future Perspective Research into ne
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56. Yonemura Y, Endo Y, Fujita H, K
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126. López-Boado YS, Wilson CL, Ho
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Proteins and Peptides- Reemergence
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Figure 3: Schematic representation
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Figure 6: Different mechanisms of c
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[30,33]. Clinical trials with autoi
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Therapeutic Proteins Figure 9: Mole
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Coming years will witness increasin
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Advances in Protein Chemistry Chapt
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purification methodology and laid t
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An overview of various purification
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