3SDS

Crystal structure of a mitochondrial ornithine carbamoyltransferase from Coccidioides immitis

Summary for 3SDS

• Entry DOI: 10.2210/pdb3sds/pdb
• Descriptor: Ornithine carbamoyltransferase, mitochondrial, CHLORIDE ION (3 entities in total)
• Functional Keywords: structural genomics, seattle structural genomics center for infectious disease, ssgcid, valley fever, coccidioidomycosis, otcase, mitochondrial, pathogenic fungus, dust-borne pathogen, carbamoyl phosphate, l-ornithine, l-citrulline, amino acid biosynthesis, transferase
• Biological source: Coccidioides immitis (Valley fever fungus)
• Cellular location: Mitochondrion matrix (By similarity): P0CL21
• Total number of polymer chains: 3
• Total formula weight: 115252.87

Authors

Edwards, T.E.,Abendroth, J.,Seattle Structural Genomics Center for Infectious Disease (SSGCID) (deposition date: 2011-06-09, release date: 2011-06-22, Last modification date: 2024-02-28)

Primary citation

Hewitt, S.N.,Choi, R.,Kelley, A.,Crowther, G.J.,Napuli, A.J.,Van Voorhis, W.C.
Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline.
Acta Crystallogr.,Sect.F, 67:1006-1009, 2011

PubMed Abstract: Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies.

PubMed: 21904041
DOI: 10.1107/S1744309111022159

Experimental method

X-RAY DIFFRACTION (2.8 Å)