Preparation of nitrophorin 7(Δ1-3) from Rhodnius prolixus without start-methionine using recombinant expression in Escherichia coli.
The heterologous recombinant expression of proteins in Escherichia coli without start-methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB-nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl2 gradient.
Max-Planck-Institut für Chemische Energiekonversion, D-45470 Mülheim an der Ruhr, Germany.
Carmen
C
Risse
Max-Planck-Institut für Chemische Energiekonversion, D-45470 Mülheim an der Ruhr, Germany.
Johanna J
JJ
Taing
Max-Planck-Institut für Chemische Energiekonversion, D-45470 Mülheim an der Ruhr, Germany; Fakultät für Chemie und Biochemie, Ruhr-Universität Bochum, D-44801 Bochum, Germany. Electronic address: markus.knipp@cec.mpg.de.
Markus
M
Knipp
D000818:Animals; D002852:Chromatography, Ion Exchange; D018387:Codon, Initiator; D004926:Escherichia coli / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012239:Rhodnius / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018387:Codon, Initiator; D006420:Hemeproteins; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D008715:Methionine; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D019791:Guanidine
Chelating Sepharose chromatography; Nitrophorin; Periplasm; Recombinant expression; Start–methionine