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Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties
Author(s) -
Botvinovskaya Alina V.,
Kostrikina Irina A.,
Buneva Valenti.,
Nevinsky Georgy A.
Publication year - 2013
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2286
Subject(s) - ethylenediaminetetraacetic acid , recombinant dna , immunoglobulin light chain , dna , chemistry , monoclonal antibody , affinity chromatography , microbiology and biotechnology , size exclusion chromatography , chromatography , chelation , biochemistry , antibody , biology , enzyme , organic chemistry , immunology , gene
An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA‐cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS‐PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg 2+ , Mn 2+ , Ni 2+ , Ca 2+ , Zn 2+ , and Co 2+ was individual for each MLCh preparation. K + and Na + inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis–Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA ( K m = 3–9 nM) and demonstrating high k cat values (3.4–6.9 min −1 ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases. Copyright © 2013 John Wiley & Sons, Ltd.