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Tag: CD

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Biochemistry. 2014  Nov 4;53(43):6776-85. doi: 10.1021/bi500861x. Epub 2014 Oct 23

Protein Engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.

Abstract

NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-ĸB pathway. Rational drug design targeting the IKK binding site on NEMO would benefit from structural insight, but to date the structure determination of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled-coil at the N-terminus, C-terminus or both ends of NEMO(44-111) present high thermal stability and cooperative melting, and most importantly restore IKKß binding affinity. We examined the consequences on structural content and stability by circular dichoism and nuclear magnetic resonance and measured binding affinity of each construct for IKKβ(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method to engineer short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile the rescuing of the binding affinity implies that a pre-ordered IKK-binding region of NEMO is compatible with IKK binding and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.

http://www.ncbi.nlm.nih.gov/pubmed/25286246

C-terminal coiled-coil adaptor fused to NEMO
C-terminal coiled-coil adaptor fused to NEMO

 

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Recombinant production of TEV cleaved human parathyroid hormone.

J Pept Sci. 2013 Aug;19(8):504-10. doi: 10.1002/psc.2528. Epub 2013 Jun 23.

Abstract

The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1-34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of (15) N-enriched human parathyroid hormone, (15) N PTH(1-34), generated via a plasmid vector that gives reasonable yield, low-cost protease cleavage (leaving the native N-terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS.

psc2528-fig-0009