Enhanced Expression and Purification Strategy for Recombinant Bacterially-Expressed Human Hexokinase II

Fazia Adyani Ahmad Fuad,Suriyea Tanbin

doi:10.20884/1.jm.2021.16.1.709

Abstract

Dengue virus hijacks the host cellular mechanism to propagate and survive during viral infection, in which the central carbon mechanism plays a crucial role to upregulate DENV infection through the increase of human hexokinase II (HKII) activity. Since the enzyme governs the glycolytic pathway, it has potentials as a target for anti-dengue (DENV) drug development. In this study, the production of human hexokinase II protein has been enhanced by using bacterial system for anti-dengue therapeutic purpose. The HKII gene was cloned into pET28b vector and transformed into the E. coli strain BL21 (DE3) for HKII expression. In order to obtain soluble recombinant HKII in an active form, we optimized protein expression under specific conditions at 18°C for 19 hours using Terrific Broth media, in the presence of 0.5 mM isopropyl-2-D-thiogalactopyranoside (IPTG). The pET28b-HKII construct expressed in BL21(DE3) system exhibited adequate protein expression, thus, this construct was subsequently proceeded to purification process. The expressed protein was purified to homogeneity by a combination of Immobilized Metal Ion Affinity Chromatography (IMAC)and size exclusion chromatography (SEC), resulting in pure, active bacterially-expressed HKII with a specific activity of 56. 67U.mg-1. The amount of HKII obtained from 2 L culture is 80 mg, with a yield percentage of 10.5%. Hence in this study, human HKII has successfully been cloned and expressed as a soluble protein that can be utilized for further therapeutic studies.

Highlights

Hexokinase is a crucial enzyme that governs the glycolytic pathway of all organisms, ranging from bacteria, yeast, and plants to humans and other vertebrates
Construction of Recombinant pET28b-hexokinase II (HKII) A full-length complementary DNA (cDNA) encoding human HKII was obtained from Genscript (Accession Number: NM_000189 and Clone ID: OHu25460C)
Based on the cDNA sequence of HKII, a set of primers were designed to amplify the region corresponding to HKII, where restriction sites HindIII and BamHI were added directly to the 5’ and 3’ ends of the primers

Summary

Introduction

Hexokinase is a crucial enzyme that governs the glycolytic pathway of all organisms, ranging from bacteria, yeast, and plants to humans and other vertebrates. The four mammalian HKs (ATP: D-hexose-6phosphotransferase; EC 2.7.1.1) are designated as I, II, III, and IV. These enzymes, highly conserved in amino acid sequences, differ in molecular mass, tissue distribution, regulation, and catalytic properties. HKI-HKIII have molecular masses of ~100 kDa and a relatively high affinity for glucose, and are subject to feedback regulation by physiological levels of glucose-6-phosphate (G-6-P) (Schaftingen, 2020). HKIV, more commonly referred to as glucokinase (GCK), has a molecular mass of 50 kDa, is primarily located in the liver and pancreatic pcells, has a lower affinity for glucose and is not subject to feedback regulation by physiological levels of G-6-

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