Overexpression and purification of human XPA using a baculovirus expression system

Ingrid L. Hermanson, John Turchi

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The xeroderma pigmentosum group A protein (XPA) is an essential component of the eukaryotic nucleotide excision repair (NER) process. Recombinant human XPA was expressed in baculovirus-infected insect cells as a [His]6-tagged fusion protein. A two-column purification procedure resulted in greater than 90% purity for the recombinant protein with a final yield of 0.53 mg from 200 ml of infected cells. The recombinant protein migrated as a doublet of 44 and 42 kDa upon SDS-PAGE consistent with that observed for the native protein. XPA can interact with a number of proteins including replication protein A (RPA) which has been implicated in the initial recognition of damaged DNA. Using a modified ELISA, we demonstrate that the recombinant XPA fusion protein also forms a complex with RPA independent of DNA. The ability of XPA to bind damaged DNA was assessed in an electrophoretic mobility shift assay using globally cisplatin-damaged DNA. The results revealed a slight preference for DNA damaged with cisplatin consistent with its proposed role in the recognition of damaged DNA. The recombinant XPA fusion protein was able to complement cell-free extracts immunodepleted of XPA restoring NER-catalyzed incision of cisplatin-damaged DNA in an in vitro excision repair assay. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalProtein Expression and Purification
Volume19
Issue number1
DOIs
StatePublished - Jun 2000
Externally publishedYes

Fingerprint

Baculoviridae
Purification
DNA
Replication Protein A
DNA Repair
Cisplatin
Recombinant Fusion Proteins
His-His-His-His-His-His
Repair
Fusion reactions
Proteins
Recombinant Proteins
Xeroderma Pigmentosum Group A Protein
Assays
Nucleotides
Electrophoretic mobility
Distillation columns
Electrophoretic Mobility Shift Assay
Cell Extracts
Insects

ASJC Scopus subject areas

  • Biochemistry

Cite this

Overexpression and purification of human XPA using a baculovirus expression system. / Hermanson, Ingrid L.; Turchi, John.

In: Protein Expression and Purification, Vol. 19, No. 1, 06.2000, p. 1-11.

Research output: Contribution to journalArticle

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