High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli. High yields of fully functional holoprotein synthesis in the BLi5 E. coli strain.
Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity. Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide. Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein. The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose. The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks. UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size. Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1.
León
Ruth Gretchen
RG
Department of Chemistry, University of Puerto Rico Río Piedras Campus, San Juan, PR.
Munier-Lehmann
Hélène
H
Barzu
Octavian
O
Baudin-Creuza
Véronique
V
Pietri
Ruth
R
López-Garriga
Juan
J
Cadilla
Carmen L
CL
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006862:Hydrogen Sulfide / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; C026236:hemoglobin I, non-mammalian; D006862:Hydrogen Sulfide