PMID: 9194171Apr 1, 1997Paper

Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin

Protein Engineering
S KimS Anderson

Abstract

The M90V mutant of guinea pig alpha-lactalbumin (gpLA) was expressed intracellularly in Escherichia coli using a gpLA gene fusion to the IgG-binding ('Z') domain coding sequences of staphylococcal protein A. The fusion protein was expressed as an inclusion body, then purified and refolded in vitro; CNBr cleavage of the fusion polypeptide yielded native alpha-lactalbumin. The recombinant M90V gpLA was virtually identical with natural gpLA with respect to its ability to stimulate lactose synthesis by galactosyl transferase and the recombinant and natural molecules also exhibited similar circular dichroism spectra, thermal melting profiles and NMR spectra. However, modest perturbations in the chemical shifts of amide protons in the C-helix residues, attributable to the Met-->Val mutation, were observed. In defined media, this expression system enabled the production of highly-enriched 15N- and 13C, 15N-labeled gpLA. Use of this material will allow the solution conformations of the native and molten globule states of gpLA to be characterized by high-resolution multidimensional NMR.

Citations

Oct 7, 1998·Protein Science : a Publication of the Protein Society·S Kim, J Baum
Jul 17, 1998·Current Opinion in Biotechnology·E D B Clark
Jul 28, 2015·Macromolecular Bioscience·Raul MachadoMargarida Casal
Apr 24, 2007·Journal of Chromatography. a·Steven S-S WangHwai-Shen Liu

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