Evaluation of culture media and induction conditions as a strategy for optimising soluble human Hexokinase Isoform 2 Expression in Escherichia coli BL21 StarTM (DE3)

Hexokinase isoform 2 (HK2) is a rate-limiting enzyme that catalyses the phosphorylation of glucose into glucose-6-phosphate (G6P) in the first step of glycolysis. In cancer cells, HK2 is highly expressed to meet the increased energy demand for rapid proliferation. This overexpression makes HK2 an attractive target fordrugdevelopment. Nevertheless, getting a pure, soluble recombinant HK2 to study the effects of certain compounds on this enzyme is a challenge due to the large size of the HK2 protein, ~102 kDa. The goal of this study is to optimise recombinant HK2 expression in Escherichia coli BL21 StarTM(DE3) through analysing distinct culture media and induction parameters to improve its expression for further production. Three different culture media, Luria-Bertani (LB) Broth Miller, Terrific Broth (TB), and Super Broth (SB), were evaluated in 100 ml and 500 ml. Slow growth was observed in SB media, where it took 3 hours to reach an OD600 of 0.4 in a 5% reinoculation culture. Upon induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 20 hours at 16-18 ̊C, no detectable HK2 was present in the soluble fraction of SB media. Meanwhile, TB supported rapid growth but yielded a smaller amount of soluble HK2 compared to LB. In a 100 ml culture, LB and TB produced soluble HK2, with TB having a low expression compared to LB. At the 500 ml scale-up, TB yielded insoluble protein, while both soluble and insoluble protein were detected in the LB culture, yet no detectable protein was expressed in eithersoluble or insoluble fraction of SB media. LB was then chosen as the subsequent medium for testing different IPTG concentrations (0.1 mM, 0.5 mM, and 1.0 mM). Upon investigation, the HK2 band is only visible in 0.1 mM IPTG induction, both in the soluble and inclusion body fractions, while other concentrations produced more insoluble fractions. Compared to prior research in BL21(DE3), this study demonstrates that BL21 StarTM (DE3) promotes enhanced soluble HK2 expressionin LB media upon induction of 0.1 mM IPTGduring low-temperature induction (16-18°C, 20 h).This optimisation strategy can facilitate further HK2-related drug discovery and therapeutic research