Recombinant hemoglobin II from Lucina pectinata: a large-scale method for hemeprotein expression in E. coli.
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.
Ramos
Cacimar
C
Department of Chemistry, University of Puerto Rico, Mayagüez Campus, PO BOX 9019, Mayagüez, PR 00681-9019, USA.
Pietri
Ruth
R
Lorenzo
Wilmarie
W
Roman
Elddie
E
Granell
Laura B
LB
Cadilla
Carmen L
CL
López-Garriga
Juan
J
D000818:Animals; D001665:Binding Sites; D049872:Bivalvia / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D006420:Hemeproteins; D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; D010100:Oxygen