Recombinant Hemoglobin II from Lucina Pectinata: a Large-scale Method for Hemeprotein Expression in E. Coli

Overview

Journal Protein J

Publisher Springer

Specialty Biochemistry

Date 2010 Mar 12

PMID 20221789

Citations 2

Authors

Cacimar Ramos

Ruth Pietri

Wilmarie Lorenzo

Elddie Roman

Laura B Granell

Carmen L Cadilla

Juan Lopez-Garriga

Affiliations

Soon will be listed here.

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.

Citing Articles

Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.

Diaz-Ayala R, Moya-Rodriguez A, Pietri R, Cadilla C, Lopez-Garriga J Mol Biotechnol. 2015; 57(11-12):1050-62.

PMID: 26482241 PMC: 4937220. DOI: 10.1007/s12033-015-9896-8.

Structural determinants for the formation of sulfhemeprotein complexes.

Roman-Morales E, Pietri R, Ramos-Santana B, Vinogradov S, Lewis-Ballester A, Lopez-Garriga J Biochem Biophys Res Commun. 2010; 400(4):489-92.

PMID: 20732304 PMC: 2992849. DOI: 10.1016/j.bbrc.2010.08.068.

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