PROCESS FOR PRODUCING TEMPLATE DNA AND PROCESS FOR PRODUCINGPROTEIN IN CELL-FREE PROTEIN SYNTHESIS SYSTEM WITH THE USE OF THE SAME

摘要

A process for producing a template DNA for protein synthesis which involves the step of amplifying a linear double-stranded DNA by a polymerase chain reaction (PCR) with the use of a reaction solution containing a first double - stranded DNA fragment containing a sequence encoding a protein or a part thereof, a second double-stranded DNA fragment containing a sequence overlapping the 5~-terminal domain of the first DNA fragment, a third double - stranded DNA fragment containing a sequence overlapping the 3~-terminal doma in of the first DNA fragment, a sense primer annealing with the 5~-terminal domain of the second DNA fragment and an antisense primer annealing with the 3~-terminal domain of the third DNA fragment, characterized in that the seco nd DNA fragment contains a sequence controlling gene transcription and translation and the concentrations of the second DNA fragment and the third DNA fragment in the reaction solution each ranges from 5 to 2500 pmol/L. By using this process, a template DNA for expressing and purifying a protein ca n be efficiently prepared.