Creation of a ribonuclease abzyme through site-directed mutagenesis

Fletcher, Michael C.; Kuderová, Alena; Cygler, Mirosław; Lee, Jeremy S.

doi:10.1038/3519

Cited by 21 publications

(11 citation statements)

References 30 publications

(27 reference statements)

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“…Rabbit RNase pIgG active centers likely contain only one residue of histidine, sufficient only for the catalysis of the first step of RNA hydrolysis. This suggestion is in agreement with a fact that introduction of one histidine residue into the variable part of light chain of the monoclonal antibody Jel 103 specifically interacting with poly(rI) converts it into an abzyme that hydrolyzes poly(rI) (Fletcher et al, 1998). In addition, one histidine residue together with an Me 2þ ion are involved in DNA cleavage by mammalian DNase I (Suck, 1994).…”

Section: Discussionsupporting

confidence: 75%

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Krasnorutskii

Buneva

Nevinsky

2008

J of Molecular Recognition

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Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.

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Section: Discussionsupporting

confidence: 75%

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Krasnorutskii

Buneva

Nevinsky

2008

J of Molecular Recognition

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“…Because of its potential for industrial applications, CalB has been an attractive target for enzyme engineering, and a number of amino acid residues affecting the function of CalB have been identified using such techniques as directed evolution7, 8 circular permutation,11 random or site‐directed mutagenesis 13, 18…”

Section: Resultsmentioning

confidence: 99%

Cell‐free synthesis and multifold screening of Candida antarctica lipase B (CalB) variants after combinatorial mutagenesis of hot spots

Park

Kwon

Song

et al. 2010

Biotechnology Progress

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We have developed a strategy for rapid and combinatorial optimization of the hot spot residues of enzymes. After combinatorial randomization of target locations in the Candida antarctica lipase B (CalB) gene, the individual variant genes isolated in the E.coli cells were expressed in the cell-free protein synthesis system to analyze different parameters of the resulting CalB variants. The enzymatic assays for the hydrolysis of para-nitrophenyl-ester (pNP-ester) and triglyceride, synthesis of wax ester, and thermal stability of the variant enzymes were carried out simultaneously in 96-well microtiter plates. From the 1,000 variant genes tested in each assay, we were able to identify a series of the variant enzymes having markedly improved hydrolytic, synthetic activity, or thermal stability. The improved traits of the cell-free selected CalB variants were well reproduced when the corresponding genes were expressed in Pichia pastoris. Therefore, we expect that the proposed strategy of cell-free expression screening can serve as a viable option for rapid and precise tuning of enzyme molecules, not only for analytical purposes but also for industrial applications through large scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

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“…variable part of the light chain of Jel-103 monoclonal antibody specifically interacting with poly(rI) converts itself into an abzyme that hydrolyzes poly(rI) (Fletcher et al, 1998 (Paul et al, 1989;Gololobov et al, 1995;Nevinsky and Buneva, 2002;Nevinsky et al, 2002;Buneva, 2003, 2005; and references therein). The affinity of rabbit IgGs for scDNA (in terms of K m values) corresponds to the typical affinity of Abs for antigens; the K m value (12-21 nM) for scDNA is comparable with the K m value of IgGs from SLE patients for plasmid scDNA (K m ¼ 43-92 nM) and is about three orders of magnitude higher than that of bovine DNase I (K m ¼ 46-58 mM) (Gololobov et al, 1995).…”

Section: Discussionmentioning

confidence: 96%

Antibodies against RNA hydrolyze RNA and DNA

Krasnorutskii

Buneva

Nevinsky

2008

J of Molecular Recognition

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Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins.

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Creation of a ribonuclease abzyme through site-directed mutagenesis

Cited by 21 publications

References 30 publications

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Cell‐free synthesis and multifold screening of Candida antarctica lipase B (CalB) variants after combinatorial mutagenesis of hot spots

Antibodies against RNA hydrolyze RNA and DNA

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