Plant Heat Shock Proteins Are More Effective in Enhancing Recombinant Alcohol Dehydrogenase Activity than Bacterial Ones In Vitro

Abstract

Background: Recombinant proteins produced in the cell factories are used in biological research, pharmaceutical production, and biochemical and agricultural applications. Molecular chaperones, such as heat shock proteins (Hsps), are co-expressed with recombinant proteins to enhance their yield, stability, and activity. When Escherichia coli (E. coli) is used as a cell factory, E. coli Hsps are the frequently used co-expression partners.

Objectives: We examined if there are differences in the molecular chaperone activities of plant and bacterial Hsps on recombinant protein activity. We compared the effects of the Hsps from carrot (Daucus carota) and E. coli on enhancing the recombinant alcohol dehydrogenase (ADH) activity and solubility under normal and heat conditions in vitro.

Materials and methods: His-tagged carrot Hsps (DcHsp17.7 and DcHsp70), E. coli Hsps (IbpA, IbpB, and DnaK), and ADH from a thermophile Geobacillus stearothermophilus were individually cloned in a pET11a or a pET26b vector, introduced into E. coli BL21(DE3), and expressed by isopropyl β-D-1-thiogalactopyranoside treatment (0.5 mM, 16 °C , 20 h). The recombinant proteins were purified using Ni-NTA affinity chromatography and resolved in SDS-PAGE (17%). The recombinant ADH was treated with the individual Hsps or in combination, and the enzyme activity was examined by measuring the NADH product levels at O.D.₃₄₀.

Results: The recombinant ADH was expressed at high levels in E. coli and very thermotolerant when the purified enzyme reacted (up to 70 °C). All five Hsps enhanced the ADH activity under normal and heat conditions in vitro, compared to the control. DcHsp17.7 and DcHsp70 were the most effective for improving the enzyme activity by up to 13.0- and 11.6-fold, respectively, followed by IbpA (8.4-fold), DnaK (6.5-fold), and IbpB (3.4-fold), at 37 °C . Combined incubation of DcHsp17.7-DcHsp70 and DcHsp17.7-DnaK further enhanced the ADH activity by 13.8 and 14.2-fold, respectively. DcHsp70 effectively enhanced ADH's solubility at 37 °C in vitro.

Conclusion: Our results suggest that plant Hsps can enhance recombinant protein activity, such as ADH, more effectively than their bacterial counterparts. Identifying effective molecular chaperones in the bacterial and eukaryotic domains will help enhance the production of recombinant proteins in E. coli.

Keywords: Enzyme activity; Molecular chaperone; Recombinant protein; Daucus carota; Escherichia coli.

Figure 1

Thermotolerance of the recombinant ADH. A) Purified recombinant ADH. C: E. coli BL21; ADH: Recombinant ADH expressed in transgenic E. coli BL21 constitutively expressing DcHsp70. B) Recombinant ADH activity at elevated temperatures. It was incubated at the indicated temperatures (37 ~ 60 °C) for up to 4 h, and the amount of the reaction product NADH was measured using spectrophotometry at 340 nm. The recombinant ADH levels were compared using a t-test (*p < 0.05). The recombinant ADH activities were compared using ANOVA (p < 0.05).

Figure 2

Purified recombinant Hsps. A) Purified sHsps, DcHsp17.7, IbpA, and IbpB. B) Purified Hsp70s, DcHsp70 and DnaK. The statistical comparison was performed using a t-test (ns: non-significance, *p < 0.05, and ****p < 0.0001).

Figure 3

Enhanced recombinant ADH activity by Hsp in vitro. Purified recombinant ADH (120 nM) was incubated with Hsps, DcHsp17.7, IbpA, IbpB, DcHsp70, and DnaK (up to 1200 nM) at A) 37 °C and B) 50 °C. The enzyme activity was analyzed by measuring the amount of product NADH at O.D.₃₄₀. The statistical comparison was performed using ANOVA (p < 0.05).

Figure 4

Enhanced recombinant ADH activity by combined treatment of two different classes of Hsps in vitro. Purified recombinant ADH (120 nM) was co-incubated with a sHsp (600 nM) and a Hsp70 (up to 300 nM) in the combinations of DcHsp17.7-DcHsp70, DcHsp17.7-DnaK, IbpA-DcHsp70, IbpA-DnaK, IbpB-DcHsp70, and IbpB-DnaK at A) 37 °C and B) 50 °C. The enzyme activity was analyzed by measuring the amount of product NADH at O.D.₃₄₀. The statistical comparison was performed using ANOVA (p < 0.05).

Figure 5

Recombinant ADH solubility in the presence of Hsps in vitro. Purified ADH (240 nM) was incubated with Hsps (600 ~ 1200 nM) [A) DcHsp17.7, B) DcHsp70, and C) DnaK] at 50 °C for 90 min, followed by centrifugation to obtain soluble and insoluble fractions. Proteins were resolved in SDS-PAGE (17%). C: ADH; BC: ADH treated with increasing amounts of Hsps. The statistical comparison was performed using ANOVA (p < 0.05).