Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation
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Published on: October 16, 2018
Amit S Kulkarni1, Nutan Khalap, Vasudha P Joshi
1Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India.
Abstract:
UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.
