Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones

Abstract

Background: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown.

Methods: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics.

Results: The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined.

Conclusion: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

Keywords: Type 2 diabetes mellitus; glycerol kinase; molecular chaperones; sarkosyl; solubilising agents.

Figure 1. Alignment of amino acids sequences of GK

The amino acids sequences of GK were accessed from NCBI (https://www.ncbi.nlm.nih.gov/) and aligned using CLUSTAL O (1.2.4) multiple sequence alignment program (https://www.ebi.ac.uk/Tools/msa/clustalo): GenBank accession no. CAA55365.1 (Homo sapiens), AAC52824.1 (Mus musculus), NP_001108056.1 (Danio rerio), GFP68323.1 (Saccharomyces cerevisiae), NP_494721.1 (Caenorhabditis elegans), BAI79241.1 (Trypanosoma brucei gambiense) and EDV66601.1 (Escherichia coli). The amino acids in the rectangles are involved in the active sites of GK; interaction with glycerol are marked as ‘a’, ADP as ‘b’ and with phosphate ion as ‘c’. ‘*’ indicates as identical amino acid residues; ‘:’ as conserved residues; ‘.’ as semi conserved residues; ‘-’ as absence of amino acid in the sequence.

Figure 2. Effect of IPTG concentrations, temperatures, and induction periods on His-GK solubility

(A) His-GK was induced in E. coli (BL21) with various concentrations of IPTG overnight at 37°C, and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (uninduced control); Lane 3 - Supernatant (uninduced control); Lane 4 - Pellet (1.0 mM IPTG); Lane 5 - Supernatant (1.0 mM IPTG); Lane 6 - Pellet (0.5 mM IPTG); Lane 7 - Supernatant (0.5 mM IPTG); Lane 8 - Pellet (0.2 mM IPTG); Lane 9 - Supernatant (0.2 mM IPTG). (B) Expression of the recombinant His-GK at different temperatures and analysis of the proteins by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (16°C); Lane 3 - Supernatant (16°C); Lane 4 - Pellet (20°C); Lane 5 - Supernatant (20°C); Lane 6 - Pellet (24°C); Lane 7 - Supernatant (24°C); Lane 8 - Pellet (37°C); Lane 9 - Supernatant (37°C). (C) Effect of induction time periods on the His-GK expression. His-GK was induced with 0.5 IPTG at 16°C at varying intervals of time, and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (2 h); Lane 3 - Supernatant (2 h); Lane 4 - Pellet (4 h); Lane 5 - Supernatant (4 h); Lane 6 - Pellet (6 h); Lane 7 - Supernatant (6 h); Lane 8 - Pellet (16 h); Lane 9 - Supernatant (16 h).

Figure 3. Effect of culture media and additives on His-GK solubility

(A) The culture was grown in TB media with different concentrations of glycylglycine (Gly-Gly) and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (No Gly-Gly); Lane 3 - Supernatant (No Gly-Gly); Lane 4 - Pellet (10 mM Gly-Gly); Lane 5 - Supernatant (10 mM Gly-Gly); Lane 6 - Pellet (500 mM Gly-Gly); Lane 7 - Supernatant (500 mM Gly-Gly). (B) Analysis of His-GK expression in auto-induction media with 1% glycerol. Lane 1 - Marker; Lane 2 - Pellet (1% glycerol); Lane 3 - Supernatant (1% glycerol). (C) Analysis of His-GK expression in the presence of various concentrations of ethanol. Lane 1 - Marker; Lane 2 - Pellet (3% EtOH); Lane 3 - Supernatant (3% EtOH); Lane 4 - Pellet (5% EtOH); Lane 5 - Supernatant (5% EtOH).

Figure 4. Effect of solubilizing agents on His-GK solubility

(A) The IBs were dissolved in 1% CHAPS and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (control); Lane 3 - Supernatant (control); Lane 4 - Pellet (1% CHAPS); Lane 5 - Supernatant (1% CHAPS). (B) Arginine, CTAB and DMSO were used in separate experiments and the proteins were resolved by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Original pellet; Lane 3 - Original supernatant; Lane 4 - Pellet (50 mM arginine); Lane 5 - Supernatant (50 mM arginine); Lane 6 - Pellet (0.5% CTAB); Lane 7 - Supernatant (0.5% CTAB); Lane 8 - Pellet (5% DMSO); Lane 9 - Supernatant (5% DMSO). (C) GdnHCl, SDS and urea were used to dissolve IBs and the proteins were resolved by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Original pellet; Lane 3 - Original supernatant; Lane 4 - Pellet (2 M GdnHCl); Lane 5 - Supernatant (2 M GdnHCl); Lane 6 - Pellet (1% SDS); Lane 7 - Supernatant (1% SDS); Lane 8 - Pellet (2 M urea); Lane 9 - Supernatant (2 M urea). (D) 8 M urea was used to denature the protein and the fractions were analysed by 10% SDS-PAGE. Lane 1 - Original pellet; Lane 2 - Original supernatant; Lane 3 - Marker; Lane 4 - Pellet (8 M urea); Lane 5 - Supernatant (8 M urea).

Figure 5. Purification of His-GK solubilized with sarkosyl and co-expressed with pKJE7

(A) The IBs were washed and resuspended in various concentrations of sarkosyl overnight at 4°C as described in Materials and methods, and the proteins resolved by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet (no sarkosyl); Lane 3 - Supernatant (no sarkosyl); Lane 4 - Pellet (0.1% Sarkosyl); Lane 5 - Supernatant (0.1% Sarkosyl); Lane 6 - Pellet (0.2% Sarkosyl); Lane 7 - Supernatant (0.2% Sarkosyl); Lane 8 - Pellet (1% Sarkosyl); Lane 9 - Supernatant (1% Sarkosyl). (B) The supernatant, after sarkosyl (1%) treatment, was purified using HisTrap HP column and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Marker; Lane 3 - Pellet (control); Lane 4 - Supernatant (control); Lane 5 - Pellet (1% sarkosyl); Lane 6 - Supernatant (1% sarkosyl); Lane 7 - Purified His-GK. (C) His-GK was co-expressed with chaperone plasmids and the proteins were analysed by 10% SDS-PAGE. Lane 1 - Pellet (pG-Tf2); Lane 2 - Supernatant (pG-Tf2); Lane 3 - Pellet (pTf16); Lane 4 - Supernatant (pTf16); Lane 5 - Pellet (pG-KJE8); Lane 6 - Supernatant (pG-KJE8); Lane 7 - Pellet (pGro7); Lane 8 - Supernatant (pGro7); Lane 9 - Pellet (pKJE7); Lane 10 - Supernatant (pKJE7). (D) Purification of His-GK co-expressed with pKJE7 and analysis of the proteins by 10% SDS-PAGE. Lane 1 - Marker; Lane 2 - Pellet; Lane 3 - Supernatant; Lane 4 - Purified His-GK.

Figure 6. Effect of buffers and pH on His-GK activity

The purified enzyme from the SEC column was used for enzyme kinetics as described in Materials and methods. (A) Effect of various buffers (50 mM) on the specific activity of His-GK. (B) Effect of pH (6.5–10.0) on the specific activity of His-GK using TEA buffer.

Figure 7. Effect of metal ions on His-GK activity

The ideal metal ion for His-GK activity was also determined as described in Material and Methods. (A) Effect of various metal ions (KCl, LiCl, NaCl, MgCl₂ and CaCl₂) on the specific activity of His-GK. (B) Effect of various concentrations of KCl (10–140 mM) on the specific activity of His-GK. (C) Effect of different concentrations of Mg²⁺ (0.6–10 mM) on the specific activity of His-GK.

Figure 8. Effect of different concentrations of PEP on His-GK activity

Different concentrations of PEP (0.1–2.0 mM) were used to determine the Vmax and Km of His-GK for PEP. (A) Lineweaver-Burk Plot showing Vmax and Km of PEP for His-GK activity. (B) Michaelis-Menten plot of His-GK in presence of various concentrations of PEP. (C) Eadie–Hofstee plot for PEP with Vmax=1.585 U/mg protein and Km=0.223 mM (R²=0.967).

Figure 9. Effect of different concentrations of glycerol on His-GK activity

Different concentrations of glycerol (1–50 µM) were used to determine the Vmax and Km of His-GK for glycerol. (A) Lineweaver–Burk plot showing Vmax and Km of glycerol for His-GK activity. (B) Michaelis–Menten plot of His-GK in presence of various concentrations of glycerol. (C) Eadie–Hofstee plot for glycerol in His-GK with Vmax = 1.548 U/mg protein and Km = 5.022 µM (R² = 0.927).

Figure 10. Effect of different concentrations of ATP on His-GK activity

Different concentrations of ATP (0.2 to 2.0 mM) were used to determine the Vmax and Km of His-GK for ATP (A) Lineweaver–Burk plot showing Vmax and Km of ATP for His-GK activity. (B) Michaelis–Menten plot of His-GK in presence of various concentrations of ATP. (C) Eadie–Hofstee plot for ATP with Vmax=1.130 U/mg protein and Km=0.767 mM (R²=0.928).