Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli

OsNifU1A is a NitU-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-Iike 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 OsNifLJ1A domain II fusion proteins. One construct has a (H'S)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-HCI. About 36% of the total (HiS)(6)/ OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCI was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(H'S)6 fusion protein was about 60% of the total cell lysate. About 2 mg of N-15-labeled OsNifUlA domain II was purified for NMR spectral studies. Examination of the OsNifUlA domain II H-1-N-15 HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of N-15-labeled OsNifUlA domain II in quantities sufficient for heteronuclear NMR solution structure studies. (c) 2005 Elsevier Inc. All rights reserved.