Catalytic Antibodies as Designer Proteases and Esterases

Tanaka, Fujie

doi:10.1021/cr010180a

Cited by 78 publications

(44 citation statements)

References 130 publications

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“…) and HindIII-NNK6-SfiI-b (5′-GAGGAGGA-GAAGCTTGTA-(MNN) 6 GGCCGCCT-GGGCCACGGT-3′), where M ) A or C and N ) A, C, G, or T. The PCR conditions were as follows: 1 µg of each primer, 100 ng of the template, 2.5 mM each dNTP, and 2.5 units of Taq polymerase in a total volume of 100 µL, with a program of 94°C for 30 s; 94°C for 15 s, 56°C for 15 s, 72°C for 1 min (25 times); and 72°C for 5 min. The PCR product was digested with EcoRI-HindIII, and the digested fragment was ligated to EcoRI-HindIII-digested construct A to give the (NNK)6-YLK library in pComb3X.…”

Section: T T T T G a A T T C A G G A G G A A T T T A A A A T -G A A Amentioning

confidence: 99%

“…Oligonucleotides YLK18-f (5′-GAGGAGGTGGCCCAGGCGGCCTACAAGCT-TCTGAAGGAACTGCTCGCTAAACTGAAGTGGCTG-CTCCGTAAACTG-3′) (10 µg) and YLK18-NNK6-b [5′-GAGGAGGAGGCCGGCCTGGCC(MNN) 6 CAGTTTACGG-AGCAGCCA-3′] (10 µg) and 2.5 mM each dNTP were mixed with 10× NEBuffer 1 (New England Biolabs, NEB) (10 µL) and with 10× NEBuffer 2 (NEB) (10 µL) in a total volume of 196 µL, and the mixture was heated at 95°C for 2 min. After being cooled to 25°C, 20 units of DNA polymerase I, large (Klenow) (5 units/µL, 4 µL), was added to the mixture.…”

Section: Construction Of the Ykllkellaklkwllrklxxxxxx Library [Ylk-(nmentioning

confidence: 99%

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Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

2005

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Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer R-helix peptide containing lysine residues. Reactionbased selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.Generation of enzyme-like small designer peptide catalysts that achieve the proficiency of natural enzymes with desired substrate specificity is a challenging task. Designer protein catalysts have been prepared by engineering naturally occurring enzymes (1-4) and by using antibody libraries (i.e., immune diversity) and selection with designed small molecule compounds (5-7). For experimental purposes, small peptides are more useful and convenient than large proteins, as long as they provide the same function, because small peptides can be more easily prepared and their characterization is simpler. However, fundamental questions remain to be answered: Can small peptides attain the catalytic efficiency of larger protein catalysts, and how can these peptide catalysts best be developed? The folded states of enzymes are key to their catalytic activity since structure can be used productively to modify the chemical reactivity of amino acid side chains. Small peptides are often limited in their potential to catalyze reactions because of the limited ability to adopt well-defined structures. Small peptides have also demonstrated limited specificity for small substrate molecules.For small catalytic peptides (<50 amino acid residues) that function in aqueous buffer, rational design has been used for the creation of catalysts of the decarboxylation of oxaloacetic acid (8-12), ester hydrolysis (13-15), and transesterifications (13). Rational design, however, is limited in throughput. A combination of rational design and librarybased methods with reaction-based selection, which has been used for the development of antibody catalysts (7), may be an attractive approach to small peptide catalyst development (16,17). Here we have explored strategies for the creation of small designer enzymes. We have developed small peptides...

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Section: T T T T G a A T T C A G G A G G A A T T T A A A A T -G A A Amentioning

confidence: 99%

Section: Construction Of the Ykllkellaklkwllrklxxxxxx Library [Ylk-(nmentioning

confidence: 99%

Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

2005

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“…Artificial Abzs can be obtained by immunization of animals with chemically stable analogs of transition states of chemical reactions (reviewed in Martin & Schultz, 1999;Tanaka, 2002;Tanaka and Barbas, 2002;Dias et al, 2002;Keinan, 2005). On the other hand, artificial antiidiotypic Abs can also possess catalytic activity (Barbas et al, 1997;Wentworth et al, 1998).…”

Section: The Origin Of Artificial and Natural Abzymesmentioning

confidence: 99%

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients

Nevinsky¹

2011

Understanding HIV/AIDS Management and Care - Pandemic Approaches in the 21st Century

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“…Whereas substrate affinity may be evaluated directly by searching for library members that increase in concentration upon exposure to this substrate, the transient character of the transition state of a chemical reaction makes a similar direct assessment of transition state binding impossible. However, by using a stable molecule that resembles the structural and electronic nature of the transition state (a transition state analogue (TSA); this strategy has been extensively utilized in the field of catalytic antibodies), [14] an indirect assessment of transition state binding may still be obtained. Following the two screening steps, systems may be identified for which the substrate and the TSA amplify the same species.…”

mentioning

confidence: 99%

Transient Substrate‐Induced Catalyst Formation in a Dynamic Molecular Network

et al. 2014

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In biology enzyme concentrations are continuously regulated, yet for synthetic catalytic systems such regulatory mechanisms are underdeveloped. We now report how a substrate of a chemical reaction induces the formation of its own catalyst from a dynamic molecular network. After complete conversion of the substrate, the network disassembles the catalyst. These results open up new opportunities for controlling catalysis in synthetic chemical systems.

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Catalytic Antibodies as Designer Proteases and Esterases

Cited by 78 publications

References 130 publications

Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients

Transient Substrate‐Induced Catalyst Formation in a Dynamic Molecular Network

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