A self-induction method to produce high quantities of recombinant functional flavo-leghemoglobin reductase

Urarte, Estibaliz, Auzmendi, Inigo, Rol, Selene, Ariz, Idoia, Aparicio-Tejo, Pedro, Arredondo-Peter, Raul and Moran, Jose F. (2008) A self-induction method to produce high quantities of recombinant functional flavo-leghemoglobin reductase. Methods in Enzymology, 436 411-423. doi:10.1016/S0076-6879(08)36023-6


Author Urarte, Estibaliz
Auzmendi, Inigo
Rol, Selene
Ariz, Idoia
Aparicio-Tejo, Pedro
Arredondo-Peter, Raul
Moran, Jose F.
Title A self-induction method to produce high quantities of recombinant functional flavo-leghemoglobin reductase
Journal name Methods in Enzymology   Check publisher's open access policy
ISSN 0076-6879
ISBN 9780123742773
Publication date 2008-01-01
Sub-type Critical review of research, literature review, critical commentary
DOI 10.1016/S0076-6879(08)36023-6
Volume 436
Start page 411
End page 423
Total pages 13
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Language eng
Subject 1303 Specialist Studies in Education
1312 Molecular Biology
Abstract Ferric leghemoglobin reductase (FLbR) is able to reduce ferric leghemoglobin (Lb3+) to ferrous (Lb2+) form. This reaction makes Lb functional in performing its role since only reduced hemoglobins bind O2. FLbR contains FAD as prosthetic group to perform its activity. FLbR-1 and FLbR-2 were isolated from soybean root nodules and it has been postulated that they reduce Lb3+. The existence of Lb2+ is essential for the nitrogen fixation process that occurs in legume nodules; thus, the isolation of FLbR for the study of this enzyme in the nodule physiology is of interest. However, previous methods for the production of recombinant FLbR are inefficient as yields are too low. We describe the production of a recombinant FLbR-2 from Escherichia coli BL21(DE3) by using an overexpression method based on the self-induction of the recombinant E. coli. This expression system is four times more efficient than the previous overexpression method. The quality of recombinant FLbR-2 (based on spectroscopy, SDS-PAGE, IEF, and native PAGE) is comparable to that of the previous expression system. Also, FLbR-2 is purified near to homogeneity in only few steps (in a time scale, the full process takes 3 days). The purification method involves affinity chromatography using a Ni-nitrilotriacetic acid column. Resulting rFLbR-2 showed an intense yellow color, and spectral characterization of rFLbR-2 indicated that rFLbR-2 contains flavin. Pure rFLbR-2 was incubated with soybean Lba and NADH, and time drive rates showed that rFLbR-2 efficiently reduces Lb3+.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Critical review of research, literature review, critical commentary
Collection: School of Agriculture and Food Sciences
 
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Created: Wed, 19 Nov 2014, 01:07:22 EST by Inigo Auzmendi on behalf of School of Agriculture and Food Sciences