Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosus

Abstract

Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 1008C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts.

Characterization of ATPase activity of class II chaperonin from the hyperthermophilic archaeon Pyrococcus furiosus

“…was present in all steps of chaperonin purification in our protocol. Our results are consistent with chaperonins described by others, including Tanaka et al (2005), Laksanalamai et al (2006), Furutani et al (1998), Andrä et al (1998), Iizuka et al (2001), and Kusmierczyk and Martin (2003). In the presence of Cd 2?…”

Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus

“…In the presence of PfCPN, higher concentrations of ATP resulted in the higher amounts of lysozyme enzyme activity reserved, while no protection effect or even further decrease of the lysozyme activity was observed when PfCPN was added alone without ATP. This result is consistent with the recent report by Pongpan Laksanalamai in which PfCPN could increase the stabilization of Taq DNA polymerase at high temperature in ATP dependence (Laksanalamai et al 2006), although in their report PfCPN alone showed slightly thermal protection effect on Taq polymerase.…”

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

“…These results are consistent with the prior work of Laksanalamai et al (2001Laksanalamai et al ( , 2006, who found that Pfu-sHSP could improve thermostabilty of Taq DNA polymerase and protected bovine glutamate dehydrogenase by aggregation prevention.…”

“…Small HSPs can effectively prevent other proteins from heat-induced aggregation but only a few reports have described thermal protection on enzyme activities. Laksanalamai et al (2006) reported that Pfu-sHSP could enhance the Taq DNA polymerase performance in PCR. Our experiments have confirmed their results.…”