University of Utah Research Foundation

Barry C Cole

Curtis L Atkin

Ann Pole

Arnold R Oliphant

Kevin L Knudtson

A method of purifying Mycoplasma arthritidis mitogen (MAM) to electrophoretic and sequence homogeneity is disclosed. A preparation of MAM purified according to this method was used to determine the sequence of the N-terminal 54 amino acids of MAM. A synthetic peptide consisting of amino acids 15-32 inhibited MAM-induced cell proliferation in vitro. The sequence of the N-terminal 54 amino acids was reverse translated, nucleotide probes were designed therefrom, and the MAM gene was selected from a genomic library. The MAM gene was sequenced and found to be contained on a 1107 bp DNA fragment. The primary translation product contains a 39 amino acid signal sequence and a 213 amino acid mature MAM (molecular weight 25,294). Amino acid sequence comparisons of MAM to bacterial and murine tumor virus superantigens showed regions of conservative sequence homology, including the region capable of inhibiting cell proliferation. Sequence homologies to HIV and other retrovirus proteins and to certain regulatory proteins were also detected. Strategies for blocking or immunizing against certain diseases, including autoimmune diseases, are disclosed.

Mycoplasma arthritidis T-cell mitogen

The gene encoding the superantigen Mycoplasma arthritidis T-cell mitogen (MAM) was molecularly cloned. The mam gene was modified by site-directed mutagenesis to change UGA codons, which in mycoplasmas are read as tryptophan codons instead of universal stop codons, to UGG codons such that the gene could be expressed in standard expression systems. Recombinant MAM, including extra amino acid residues at the N- and C-termini, were expressed and discovered to be biologically active. Certain amino acid substitutions in active regions of the protein also yield biologically active MAM proteins. A method of diagnosing rheumatoid arthritis using recombinant MAM protein in an ELISA assay is disclosed.

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Sandy, UT, United States of America

Barry C Cole