Trial of Efficient Protein Expression Using Thermophilic Archaea as a Host

Thermostable enzymes of (hyper)thermophiles which exhibit optimal growth at temperatures above 55℃ have considerable potential for enzyme-based technologies as robust catalyst alternatives because most of them hardly unfold under various denaturing conditions. However, some thermostable proteins from hyperthermophiles often aggregate and form inclusion complex when they are expressed in mesophilic hosts such as Escherichia coli. A hyperthermophilic archaeon, Thermococcus kodakarensis grows fast and shows high cell density in nutrient medium and genetic technics including gene knockout and complementation by plasmid. T. kodakarensis contains a transcription repressor, SurR, which binds to the SurR-binding consensus sequence (SBS, GTTn3AAC) by causing conformational change in the absence of element sulfur (S0). S0 was expected as an inducer for the expression of interest of gene in an expression system in T. kodakarensis. The aim of this research is to construct an efficient protein expression system in T. kodakarensis cells as a host. To screen the suitable promoter in T. kodakarensis expression system, four promoters Tk0560, Tk1694, Tk2289 and Tk0895, have been chosen based on reported transcriptome analysis. Each DNA fragment containing the promoter region of these genes was cloned into Escherichia coli-T. kodakarensis shuttle plasmid pTKDR-His, yielding the plasmids pTKDR-pTk0560, pTKDR-pTk1694, pTKDR-pTk2289, and pTKDR-pTk0895 (designated as pCSG-His). Next, the T. kodakarensis DAD (∆pyrF, ∆pdaD) cell harboring each plasmid was separately cultivated anaerobically at 85˚C in nutrient-rich ASW-YT medium in the presence of S0. The cell extracts were applied to western blot analysis, performed by using antibody raised against Pc-Kat (Pyrobaculum calidifontis catalase gene as a reporter gene). The four candidate promoters contain a highly conserved 8-bp sequence element (TWTWTAWR, where W is A/T and R is A/C) and the purine-base-rich BRE (B recognition element), those of which are important for promoter strength. Among them, Tk0895 promoter led to the highest expression of Pc-Kat. To develop the switching system of expression in T. kodakarensis, SBS was tried to be inserted between TATA-Box and TSS in pCSG-His, but expected plasmid was not obtained. It is speculated that the high copy number SBS inserted in the plasmid has some toxic effect on E. coli cell growth. Each DNA fragment containing Tk0306 and Tk0460, which encodes RNA helicase, was cloned into the plasmid pCSG-His, yielding the plasmids pCSG-His-Tk0306 and pCSG-His-Tk0460, respectively. T. kodakarensis transformant harboring pCSG-His-Tk0306 or pCSG-His-Tk0460 were cultivated in ASW-YT media. Purification of the recombinant Tk0306 and Tk0460 proteins from cell extracts was performed using Ni-chelate affinity chromatography. The recombinant Tk0306 and Tk0460 were successfully obtained from T. kodakarensis extracts while Tk0460 showed smaller molecular mass than speculated size in SDS-PAGE, suggesting the recombinant Tk0460 was not fully denatured for SDS-PAGE in the present experimental conditions.