Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli.
OsNifU1A is a NifU-like rice (Oryza sativa) protein, discovered recently. Its amino acid sequence is very homologous to the sequence of cyanobacterial CnfU and to the sequences of NifU C-terminal domains. Based on its sequence, OsNifU1A is probably a modular structure consisting of two CnfU-like domains, with domain I (formed by residues Leu73 to Gly153) and domain II (formed by residues Leu154 to Ser226). Domain I have a conserved Cys-X-X-Cys motif, which may function as an iron-sulfur cluster assembly scaffold. Domain II lacks a Cys-X-X-Cys motif and therefore, cannot function analogously. Other NifU-like proteins, with sequences homologous to OsNifU1A domain II, have been identified during plant genomic projects; however, the biological roles of these domains remain unknown. We successfully constructed an Escherichia coli expression system for OsNifU1A domain II that enabled us to synthesize and purify milligram quantities of protein for use in structural and functional studies. Using the Gateway system, we built DNA sequences corresponding to two OsNifU1A domain II fusion proteins. One construct has a (His)6 sequence upstream of the OsNifU1A domain II sequence; the other has an upstream thioredoxin-(His)6 sequence. Recombinant OsNifU1A domain II fusion proteins were extracted from E. coli inclusion bodies by dissolving them in 6 M guanidine-HCl. About 36% of the total (His)6/OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCl was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(His)6 fusion protein was about 60% of the total cell lysate. About 2 mg of 15N-labeled OsNifU1A domain II was purified for NMR spectral studies. Examination of the OsNifU1A domain II 1H-15N HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of 15N-labeled OsNifU1A domain II in quantities sufficient for heteronuclear NMR solution structure studies.
Katoh
Shizue
S
Biochemistry Department, National Institute of Agrobiological Science, Tsukuba, Ibaraki 305-8602, Japan.
Murata
Katsuyoshi
K
Kubota
Yoshiki
Y
Kumeta
Hiroyuki
H
Ogura
Kenji
K
Inagaki
Fuyuhiko
F
Asayama
Munehiko
M
Katoh
Etsuko
E
D004926:Escherichia coli / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D009586:Nitrogen Fixation; D019906:Nuclear Magnetic Resonance, Biomolecular; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D007506:Iron-Sulfur Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins