Purification, cloning, and expression of two closely related Trypanosoma brucei nucleic acid binding proteins.
Nucleic acid binding proteins in the trypanosomatid family are of particular interest because of several unusual molecular phenomena discovered in these organisms. We have purified two closely related proteins, p34 and p37, from the procyclic from of T. brucei using high salt extraction and single-stranded-DNA (ssDNA) agarose chromatography. Antibodies raised against the p34 protein showed crossreactivity with p37, suggesting relatedness. High performance liquid chromatography analysis and microsequencing of tryptic peptides derived from p34 and p37 showed that the primary structures of the two proteins are nearly identical. We have cloned and sequenced the two genes encoding these two proteins. Protein sequences predicted from the cDNAs confirm the relatedness of the two proteins but also indicate the presence of an 18 amino acid insertion unique to one of the two proteins as well as several minor differences resulting from single amino acid changes. Three sequence motifs have been identified in both proteins: an N-terminal alanine, proline, and lysine rich domain, one and a half internal RNA-binding domains, and a C-terminal KKDX repeat region. Both proteins preferentially bind to heterogenous RNA and ssDNA versus double-stranded DNA and homopolymers. Both recombinant proteins have been expressed in E. coli and show properties indistinguishable from those observed with native p34/p37.
Zhang
J
J
Department of Microbiology, State University of New York at Buffalo 14214, USA.
Williams
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins