title: A survey of methods for the purification of microbial flavohemoglobins.
abstract: Over the past decade, the flavohemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse organisms, including yeasts and fungi but particularly pathogenic bacteria. Flavohemoglobins--the best-characterized class of microbial globin--comprise two domains: a globin domain with a noncovalently bound heme B and a flavin domain with recognizable binding sites for FAD and NAD(P)H. Hmp was first identified in Escherichia coli and now has a clearly defined role in NO biology in that organism: its synthesis is markedly up-regulated by NO, and hmp knockout mutants of E. coli and Salmonella typhimurium are severely compromised for survival in the presence of NO in vitro and in pathogenic lifestyles. In the presence of molecular O2, Hmp catalyzes an oxygenase or denitrosylase reaction in which NO is stoichiometrically converted to nitrate ion, which is relatively innocuous. In this chapter, we present a survey of the methods used to express and purify the flavohemoglobins from diverse microorganisms and describe in more detail three methods developed and used in this laboratory for the E. coli protein. Particular problems are highlighted, particularly (a) the toxic consequences of Hmp overexpression that result from its ability to catalyze partial oxygen reduction and (b) the expression of protein with substoichiometric content of redox-active flavin and heme centers.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Megan E S
authors.initials: ME
authors.lastname: Lewis
authors.forename: Hazel A
authors.initials: HA
authors.lastname: Corker
authors.forename: Bridget
authors.initials: B
authors.lastname: Gollan
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004093:Dihydropteridine Reductase / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors; D006420:Hemeproteins / Q000235:genetics / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D011994:Recombinant Proteins; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases