ABSTRACT
Hemoglobin II from the clam L. pectinata is an O(2) reactive protein that remains oxygenated in the presence of other molecules. To determine the mechanism of ligand selection in this hemoglobin, rHbII was expressed in large quantities using an improved fermentation process. The highest protein yield was obtained by: transforming HbII into the BLi5 cells, inducing and supplementing the culture during the mid-log phase with 1 mM IPTG, 30 microg/mL hemin chloride and 1% glucose, and decreasing the temperature to 30 degrees C after induction. In addition, cell culture density was greatly enhanced by using glycerol, adding MgSO(4), supplementing the media with glucose after the glycerol was consumed and maintaining the dissolved oxygen at 35%. Under these conditions the maximum protein yield obtained was approximately 2,300 mg/L. The results indicate that rHbII is similar to the native protein. The protocol was validated with other hemoglobins, indicating that it can be extended to other hemeproteins.
Subject(s)
Bivalvia/metabolism , Escherichia coli/metabolism , Hemeproteins/metabolism , Hemoglobins/metabolism , Protein Engineering/methods , Recombinant Fusion Proteins/metabolism , Animals , Binding Sites , Bivalvia/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Hemeproteins/genetics , Hemoglobins/genetics , Models, Molecular , Oxygen/metabolism , Protein Conformation , Recombinant Fusion Proteins/geneticsABSTRACT
The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.
