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

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 (kon) and dissociation (koff) rate constants were 1.4 × 105/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.

Engineered (Lys)6-Tagged Recombinant Sulfide-Reactive Hemoglobin I for Covalent Immobilization at Multiwalled Carbon Nanotubes

“…The (His) 6 -tagged rHbI has comparable structural and functional properties to the native HbI and the (Lys) 6 -tagged rHbI. 12 The main difference between these two rHbI mutants is the histidine tag at the N-terminus instead of a lysine tag at the C-terminus. This (His) 6 -tagged rHbI has the same quantity of lysine residues, except for the presence of the (Lys) 6 -tagged at the C-terminus.…”

“…After the addition of H 2 S, formation of the Fe(III)–H 2 S derivative (see reaction 2 ) is expected, considering the large association rate constant 1.4 × 10 5 M –1 s –1 for the formation of the (Lys) 6 rHbI Fe(III)–H 2 S at neutral pH. 12 …”

“…This is in accordance with the results previously published by this group in which the (Lys) 6 -tag was used to interact electrostatically with a cation exchange resin for purification purposes. 12 There it was demonstrate that due to the high abundance of lysine exposed to the solvent in the C-terminal (polylysine tag), it is reasonable to suggest that these residues are more likely responsible for the coupling reaction between the (Lys) 6 -tagged rHbI and the activated carboxylic groups of the MWCNTs-COOH. Other authors that use polylysine tag employed similar rationalization.…”

“…We previously reported the construction, expression, and purification of this new (Lys) 6 -tagged rHbI together with its structural and functional properties. 12 The results showed that the (Lys) 6 -tagged rHbI is structurally analogous to the native HbI and to the (His) 6 -tagged rHbI protein that binds H 2 S. In addition, the results indicated that the lysine residues located in the tag are more solvent exposed (>60%) than the other three native lysine residues (<60%). Thus, the polylysine tag is most likely responsible for the interaction between the (Lys) 6 -tagged rHbI and biocompatible surfaces.…”

“…For example, Allard et al 10 and Ladavière et al 11 successfully immobilized a polylysine tagged protein by a covalent bond. Our recombinant hemoglobin I (HbI) from Lucina pectinata was engineered to contain a polylysine tag, 12 which allowed it to attach covalently on conductive and biocompatible multiwalled carbon nanotubes (CNTs). This will be a viable approach to further develop a prototype detection system for hydrogen sulfide because HbI binds H 2 S with high affinity.…”