[14] Synthesis of proteins by subtiligase

Ac, Braisted; Jk, Judice; Ja, Wells

doi:10.1016/s0076-6879(97)89053-2

Cited by 71 publications

(55 citation statements)

References 21 publications

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“…Noteworthy is the development of enzymatic ligation methods for the preparation of large polypeptides from synthetic peptide segments, with ligase enzymes specifically engineered for this purpose by the methods of molecular biology (45). Ironically, the principal obstacle to general utility of enzymatic ligation has proven to be the limited solubility of even the unprotected peptide segments, under the physiological conditions compatible with the enzymes used (46). Despite considerable efforts and some notable successes (47), such methods have not found widespread use.…”

Section: Figurementioning

confidence: 99%

Synthesis of Native Proteins by Chemical Ligation

Dawson

Kent²

2000

Annu. Rev. Biochem.

1,582

1,526

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Key Words chemical protein synthesis, thioester, protein, peptide, solid phase synthesis, polymer-supported synthesis, protein engineering s Abstract In just a few short years, the chemical ligation of unprotected peptide segments in aqueous solution has established itself as the most practical method for the total synthesis of native proteins. A wide range of proteins has been prepared. These synthetic molecules have led to the elucidation of gene function, to the discovery of novel biology, and to the determination of new three-dimensional protein structures by both NMR and X-ray crystallography. The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems. Chemical protein synthesis has also enabled the systematic development of proteins with enhanced potency and specificity as candidate therapeutic agents.

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Section: Figurementioning

confidence: 99%

Synthesis of Native Proteins by Chemical Ligation

Dawson

Kent²

2000

Annu. Rev. Biochem.

1,582

1,526

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“…However, catalysis of peptide bond formation between native protein0peptide segments by proteases in neat aqueous solution usually do not occur appreciably even in favorable situations entailing stereochemical proximity of the reacting ends, as in proteolytically nicked and thermodynamically stable noncovalent complexes of proteins, primarily because the protease-catalyzed peptide bond hydrolytic equilibria is not adequately shifted in the direction of ligation in 55.5 M water. Nonetheless, proteases catalyze the condensation of peptides in aqueous solution with the use of C-terminal active esters~Schellenberger & Jakubke, 1991; Wong & Wang, 1991;Braisted et al, 1997!. Here we report that generation of productdirected conformational traps can act as a driving force for the protease-catalyzed splicing of native peptide segments in neat aqueous solutions, thereby obviating the necessity of active esters or organic cosolvents or strong interaction between the reacting peptides.…”

Section: Discussionmentioning

confidence: 88%

“…The peptide oligomerization reaction described here suggests that V8 protease is endowed with considerable synthetic potential in aqueous solution. The enhancement of innate peptide ligation activity of V8 protease by protein engineering is conceivable in light of the successful development of engineered subtilisins as peptide ligases~Abrahmsen et al, 1991;Chang et al, 1994;Braisted et al, 1997;Atwell & Wells, 1999!. The broad pH-activity profile of V8 protease~Birktoft & Breddam, 1994!…”

Section: Discussionmentioning

confidence: 99%

“…In such cases, "trapping" of the product shifts the equilibrium to synthesis due to continuous removal of the spliced material from the chemical equilibrium Homandberg et al, 1982;Nyberg, 1988;Roy et al, 1992;Kumaran et al, 1997;Sahni et al, 1998!. The catalysis of amide bond formation by proteases proceeds as well in neat aqueous solution, but only with peptide esters as substrate. The systematic protein engineering of subtilisin by chemical modification, site-directed mutagenesis, and phage-display technology has yielded a variant enzyme, subtiligases, that can efficiently catalyze peptide ligation in aqueous solution~Nakatsuka et al, 1987;Wu & Hilvert, 1989;Abrahmsen et al, 1991;Chang et al, 1994;Braisted et al, 1997;Atwell & Wells, 1999!. The total synthesis of ribonuclease A~RNase A!…”

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confidence: 99%

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An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution

2000

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We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free a-carboxyl and a-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 8C, producing a gamut of covalent oligomers~dimer through octamer or higher! of a native protein segment TAAAKFE~S39! derived from ribonuclease A~RNAse A!. Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."

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“…The relatively dry conditions favor the reaction of the N-terminal amide with the acyl-enzyme intermediate in the active site of the protease. Probably the most effective enzymic ligation system is a modified subtilisin, called subtiligase, and its derivatives, which have significantly reduced rates of proteolysis and therefore allow reaction in aqueous media [15][16][17][18][19]. Enzymatic ligation has been used to incorporate moieties such as biotin, heavy atoms and sugars into synthetic or partially synthetic proteins.…”

Section: Chemical Synthesis and Semi-synthesis Of Proteinsmentioning

confidence: 99%

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Magliery¹

2005

MCRO

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Less than a decade ago, the ability to generate proteins with unnatural modifications was a Herculean task available only to specialty labs. Recent advances make it possible to generate reasonable quantities of protein with unnatural amino acids both in vitro and in vivo . The combination of solid-phase peptide synthesis and enzymatic or chemoselective ligation now permits construction of entirely-synthetic proteins as large as 25 kD. Incorporation of recombinant fragments (expressed protein ligation) allows unnatural modifications near protein termini in proteins of virtually any size. Site-specific modification of small quantities of protein at any position can be achieved using chemically-acylated tRNA. Microelectroporation now extends this method to cells like mammalian neurons, and combination with RNAdisplay makes unnatural proteins compatible with combinatorial methods. Widespread or residue-specific methods of amino acid replacement are especially suitable for the production of biomaterials, and bacteria have been engineered to expand the repertoire of amino acids available for this technique. Excitingly, the wholesale addition of engineered tRNAs and synthetases to bacteria and yeast now makes site-specific incorporation of unnatural amino acids possible in living cells with no chemical intervention. Methods of expanding the genetic code at the nucleic acid level, including 4-base codons and unnatural base pairs, are becoming useful for the addition of multiple amino acids to the genetic code. These recent advances in unnatural protein engineering are reviewed with an eye toward future challenges, including methods of creating nonpeptidic molecules using templated synthesis.

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[14] Synthesis of proteins by subtiligase

Cited by 71 publications

References 21 publications

Synthesis of Native Proteins by Chemical Ligation

Synthesis of Native Proteins by Chemical Ligation

An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

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