Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli.
Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.
Zuo
Zhuang
Z
Gene Discovery and Modification Laboratory, Department of Transformation Research, Pioneer Hi-Bred International, Inc. (A DuPont Company), Johnston, IA 50131, USA.
Mahajan
Pramod B
PB
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins