Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus.
A number of ferriheme proteins, termed nitrophorins (NPs), occur in the saliva of the bloodsucking insect Rhodnius prolixus ('kissing bug'), which is a vector for Chagas' disease. Nitrophorins bind the heme b cofactor in the beta-barrel of their lipocalin fold, which is further anchored through a proximal histidine-Fe(III) bond. The distal Fe(III) coordination site then binds nitric oxide (NO) for delivery into a host's tissues during blood feeding, where, upon NO release, the distal Fe(III) site acts as a histamine trap to delay the victim's immune response. Previously, four nitrophorins from R. prolixus, NP1 to NP4, have been extensively characterized. Recently, another nitrophorin, NP7, was discovered in a cDNA library derived from the same insect. Among the R. prolixus nitrophorins, NP7 was found to be unique in its ability to bind to negatively charged cell surfaces. However, the yield of functional recombinant NP7 was rather low when the established protocol for NP1-4 was followed. Here, we report on a novel expression and reconstitution method for NP7 that yields sufficient amounts of pure protein for extensive characterization (28-fold increase). This method may prove useful for the reconstitution of other proteins with a lipocalin fold.
Knipp
Markus
M
Department of Chemistry, University of Arizona, 1306 East University Boulevard, Tucson, AZ 85721-0041, USA.
Zhang
Hongjun
H
Berry
Robert E
RE
Walker
F Ann
FA
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D006420:Hemeproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006427:Hemin / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012239:Rhodnius / Q000235:genetics / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D006420:Hemeproteins; D008081:Liposomes; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D006427:Hemin