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Published on: October 2, 2012
Plant Heat Shock Proteins Are More Effective in Enhancing Recombinant Alcohol Dehydrogenase Activity than Bacterial
Minjae Jung1, Yunjin Park1, Yeh-Jin Ahn1
1Department of Biotechnology, Sangmyung University, 20 Hongjimun 2-gil, Jongno-gu, Seoul 03016, Korea.
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.
