Chemical Approaches to Protein Engineering

Offord, Robin E.

doi:10.1007/978-1-4684-5739-1_13

Cited by 7 publications

(4 citation statements)

References 68 publications

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“…The chemical synthesis ofpeptides has been important for the understanding of protein structure-function relationships and the discovery of new therapeutic agents (1). At present, stepwise solid-phase peptide synthesis (2) can efficiently prepare moderate-size peptides ofnearly 100 amino acids (3).…”

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Peptide segment ligation strategy without use of protecting groups.

Liu

Tam

1994

Proc. Natl. Acad. Sci. U.S.A.

210

161

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We describe the concept and the verification of a chemical ligation approach to the synthesis of proteins using peptide se-ents with no protecting groups and no activation of the C-tenal a-carboxyl group. This approach of three steps: (i) aldehyde introduction, in which a masked glycolaldehyde ester Is linked to the carboxyl terminus of an unprotected peptide by reverse protedlysis; (ii) ring formation, in which the umaked aldehyde reacts with the N-teinal a-amino group of the second unprotected peptide containing either a cysteine or a threonine residue to form a t ne or olidine ring at an acidic pH; and (iii) rearrangement in which O-acyl ester linkage is transferred to N-acyl amide linkage to form a peptide bond with a pseudoproline stuture at higher pH. The (4), and the semi-synthesis of proteins consisting of components from synthetic peptides with unusual amino acids and proteins obtained through recombinant technology.The conventional segment-synthesis approaches (5) usually require a maximal protection strategy which has the limitations ofpoor solubility, low coupling efficiency, and the possible danger of racemization due to the overactivation of the carboxyl group. Considerable efforts have been made to overcome these limitations through the use of partial or minimal protecting-group strategies and improvements in the activation methods (6-11). However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules. Alternative approaches to overcome the difficulty of forming amide bonds use hydrazone (12,13) or thioether and thioester (14) as surrogate bonds. The advantage of these peptide-bond replacement approaches resides in the high and specific reactivity between the two functional groups not found in amino acids to make the conjugation efficient. The disadvantage is that these linkages are substantially different from the peptide bond, which may lead to conformational distortion, and are usually not stable at either acidic (12) or basic (14) pH.We have now designed and developed a chemical ligation method by which two unprotected peptide segments can be ligated together through an amide bond. In our design, we make use of a highly regiospecific and efficient reaction to link covalently two unprotected peptides. The selectivity of this reaction would also obviate the need for any protecting groups. A peptide bond is then formed in a second step through an intramolecular rearrangement between the two closely neighboring carboxyl and amino groups. The efficiency of the first reaction would solve the slow kinetic problem for reactions between large molecules, and the second reaction, designed as a spontaneous process to form amide bonds without activating the carboxyl group, would overcome the racemization problem of the conventional segment condensation method. EXPERIMENTAL PROCEDURESGeneral. 1H NMR spectra were obtained at 300 ...

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confidence: 99%

Peptide segment ligation strategy without use of protecting groups.

Liu

Tam

1994

Proc. Natl. Acad. Sci. U.S.A.

210

161

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“…These chemical methods suffered a number of limitations, sufficient enough that their use has been relatively restricted, although of late methods to avoid many of the difficulties have evolved. For a full discussion of these developments, see Kent (2), Muir and Kent (3), Offord (4,5), and Wallace (6,7).…”

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Synergy in Protein Engineering

Woods

Guillemette

Parrish

et al. 1996

Journal of Biological Chemistry

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Semisynthesis is a chemical technique of protein en-gineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.

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“…The rebuilding of proteins from two or more fragments has been used for many years to investigate structurefunction relationships of proteins (for reviews, Chaiken, 1981; Offord, 1990). Though mutation of recombinant proteins is now a more generally suitable approach for these studies, the coupling of protein fragments remains important in three areas: firstly, for the introduction of noncoded structures (e.g.…”

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“…Though mutation of recombinant proteins is now a more generally suitable approach for these studies, the coupling of protein fragments remains important in three areas: firstly, for the introduction of noncoded structures (e.g. D-amino acids and peptide mimics; Offord, 1990); secondly, to overcome the length limitations of solid-phase peptide synthesis (Kent, 1992); and thirdly, to conjugate site-specifically various structures, as for example introducing a "reporter" group for structural studies of a protein or arming antibodies with peptidic toxins (Waldman, 1991). For such purposes, the ability of proteolytic enzymes to catalyse peptide-bond synthesis has been widely investigated with the aim of developing new methods of fragment coupling which can be achieved with great specificity and minimum use of protecting groups Jakubke et al, 1985).…”

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Construction of protein analogs by site-specific condensation of unprotected fragments

Gaertner

Rose²,

Cotton³

et al. 1992

Bioconjugate Chem.

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146

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The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.

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Chemical Approaches to Protein Engineering

Cited by 7 publications

References 68 publications

Peptide segment ligation strategy without use of protecting groups.

Peptide segment ligation strategy without use of protecting groups.

Synergy in Protein Engineering

Construction of protein analogs by site-specific condensation of unprotected fragments

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