Expression in Escherichia coli of the putative N-acetylneuraminate lyase gene (nanA) from Haemophilus influenzae: overproduction, purification, and crystallization

Protein Expression and Purification
G G LilleyL A Pearce

Abstract

The cloning and expression of the Haemophilus influenzae gene, nanA, for the putative N-acetylneuraminate lyase enzyme, also known as N-acetylneuraminic acid aldolase or sialic acid aldolase, are reported. The gene was isolated from ATCC type strain 49247 and cloned into the Escherichia coli expression vector pKKtac, which contained the strong tac promoter. Gene expression was compared with the homologous E. coli npl gene coding for the lyase. Purification protocols for the products of the nanA and npl genes are presented. Activity analysis showed that the nanA gene product is a sialic acid aldolase with more than threefold greater specific activity (6.9 IU/mg) than the enzyme from E. coli (</=2 IU/mg). A method for the provision of lyase orthorhombic crystals is reported. These crystals diffract to better than 2.0 A, which paves the way to the solution of the enzyme's three-dimensional structure.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
Nov 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·P H HirelS Blanquet
Aug 1, 1984·Journal of Biochemistry·Y UchidaT Sugimori
Jul 28, 1995·Science·R D FleischmannJ M Merrick
Oct 1, 1956·Proceedings of the National Academy of Sciences of the United States of America·R Heimer, K Meyer

❮ Previous
Next ❯

Citations

Jun 4, 2008·Applied Microbiology and Biotechnology·Yanhong LiXi Chen
Jun 9, 2000·Molecular Microbiology·E VimrS Steenbergen
Feb 15, 2011·Applied and Environmental Microbiology·Guiomar Sánchez-CarrónAlvaro Sánchez-Ferrer
May 13, 2014·Applied and Environmental Microbiology·Muireann EganDouwe van Sinderen
Jun 12, 2019·PloS One·Man Kumari GurungInger Lin U Ræder
Sep 10, 2015·Microbiology Spectrum·Brandy L Haines-MengesE Fidelma Boyd
Jul 19, 2003·Organic Letters·Jianbo ZhangPeng George Wang

❮ Previous
Next ❯

Related Concepts

Related Feeds

Biosynthetic Transformations

Biosyntheic transformtions are multi-step, enzyme-catalyzed processes where substrates are converted into more complex products in living organisms. Simple compounds are modified, converted into other compounds, or joined together to form macromolecules. Discover the latest research on biosynthetic transformations here.