Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA

Abstract

The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase, reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60 degrees C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA.

Figure 1

Differences between stabilization and activation. (A) Stabilization: relative activity of SfyI at various temperatures in the absence or presence of trehalose. (B) Activation: relative activity of DNaseI and NcoI restriction enzyme at the indicated temperatures in the absence or presence of trehalose. °C axis, temperature of incubation; % axis, relative activity (arbitrary units). Data are representative of three independent sets of experiments.

Figure 2

Improvement of full length cDNA yield by thermostabilization of RT by trehalose. (A) 5-kb cDNAs were synthesized in the presence of 0.6 M trehalose at 45°C (lane 1), 55°C (lane 2), 60°C (lane 3), and 65°C (lane 5); lane 4, synthesis at 60°C in the absence of trehalose. (B) In vitro transcripts were used as templates as follows: lanes 1–4, 5 kb; lanes 5–8: 5.5 kb; lanes 9–12: 10 kb. Lanes 1, 5, and 9: reactions without trehalose; lanes 2–4, 6–8, and 10–12: reactions with trehalose. Lanes 1, 2, 5, 6, 9, and 10: incubation at 45°C; lanes 3, 7, and 11: incubation at 60°C; lanes 4, 8, and 12: incubation with temperature cycling. Arrowheads, full length cDNAs; asterisks, truncated cDNAs. (C) RT of mouse brain mRNA: lane 1, standard optimized reaction; lane 2, reaction in the presence of trehalose at standard temperature; lane 3, reaction in absence of trehalose with the temperature-cycling program; lane 4, reaction in presence of trehalose with the temperature cycling program. M, λ-HindIII markers. Figures are representative of at least three independent experiments. Longer cDNAs synthesized in the presence of trehalose (lane 4, arrow) or under standard best conditions (lane 1, asterisks) are shown.

Figure 3

Comparison of trehalose with other sugars for enzyme stabilization or activation. In vitro transcript of 5 kb was used as substrate for cDNA synthesis at 60°C in the presence of the following reaction modifiers: lane 1, trehalose; lane 2, sucrose; lane 3, glucose; lane 4, maltose; lane 5, no addition. Arrow and asterisk, longest full length cDNA detectable in the trehalose-containing reaction and in the absence of trehalose, respectively. Data are representative of more than three independent experiments.