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

Overview

Journal Mol Biotechnol

Publisher Springer

Specialties Biotechnology
Molecular Biology

Date 2015 Oct 21

PMID 26482241

Citations 3

Authors

Ramonita Diaz-Ayala

Andres Moya-Rodriguez

Ruth Pietri

Carmen L Cadilla

Juan Lopez-Garriga

Affiliations

Soon will be listed here.

Abstract

A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.

Citing Articles

Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

Torres-Gonzalez L, Diaz-Ayala R, Vega-Olivencia C, Lopez-Garriga J Sensors (Basel). 2018; 18(12).

PMID: 30518079 PMC: 6308469. DOI: 10.3390/s18124262.

Engineered (Lys)-Tagged Recombinant Sulfide-Reactive Hemoglobin I for Covalent Immobilization at Multiwalled Carbon Nanotubes.

Diaz-Ayala R, Torres-Gonzalez L, Pietri R, Cabrera C, Lopez-Garriga J ACS Omega. 2018; 2(12):9021-9032.

PMID: 29302632 PMC: 5748273. DOI: 10.1021/acsomega.7b01500.

Chemical Biology of HS Signaling through Persulfidation.

Filipovic M, Zivanovic J, Alvarez B, Banerjee R Chem Rev. 2017; 118(3):1253-1337.

PMID: 29112440 PMC: 6029264. DOI: 10.1021/acs.chemrev.7b00205.

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