3TL6

Crystal structure of purine nucleoside phosphorylase from Entamoeba histolytica

Summary for 3TL6

• Entry DOI: 10.2210/pdb3tl6/pdb
• Descriptor: Purine nucleoside phosphorylase, SULFATE ION (3 entities in total)
• Functional Keywords: structural genomics, seattle structural genomics center for infectious disease, ssgcid, anaerobic parasitic protozoan, digestive tract cyst, maltose binding protein, mbp, fusion, pnpase, purine metabolism, nucleotide salvage pathway, transferase
• Biological source: Entamoeba histolytica
• Total number of polymer chains: 6
• Total formula weight: 160257.69

Authors

Edwards, T.E.,Clifton, M.C.,Seattle Structural Genomics Center for Infectious Disease (SSGCID) (deposition date: 2011-08-29, release date: 2011-09-07, Last modification date: 2023-09-13)

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 (2.65 Å)