Riken Corporation

Takashi Yabuki

Shigeyuki Yokoyama

Takanori Kigawa

Yoko Motoda

Mikako Shirouzu

Goushi Ishihara

Kaori Tajima

Emi Nunokawa

Provided is a method of producing a template DNA used for protein synthesis comprising a step of amplifying a linear double-stranded DNA by polymerase chain reaction (PCR), by using a reaction solution comprising a first double-stranded DNA fragment comprising a sequence coding for a protein or a portion thereof, a second double-stranded DNA fragment comprising a sequence overlapping with the 5' terminal region of the first DNA fragment, a third double-stranded DNA fragment comprising a sequence overlapping with the 3&#x2032; terminal region of the first DNA fragment, a sense primer which anneals with the 5&#x2032; terminal region of the second DNA fragment, and an anti-sense primer which anneals with the 3&#x2032; terminal region of the third DNA fragment, wherein the second DNA fragment comprises a regulatory sequence for transcription and translation of a gene, and the concentrations of the second DNA fragment and the third DNA fragment in the reaction solution each range from 5 to 2,500 pmol/L. The use of this method enables efficient production of a template DNA for expression and purification of a protein.

Process for producing template DNA and process for producing protein in cell-free protein synthesis system with the use of the same

Method of producing template DNA and method of producing protein in cell-free protein synthesis system using the same

A method for producing a protein using a cell-free protein synthesis system comprising a detergent so that the protein can be synthesized without aggregation, is provided. The protein is a protein comprising a hydrophobic region in at least a portion thereof, for example, a membrane protein or its fragment (portion). And the detergent is a mild detergent which would not denature the protein, for example, a nonionic or amphoteric ionic detergent.

Process for synthesizing protein using cell-free protein synthesis system

A method for producing a protein suitable for X-ray crystallographic analysis, in a cell-free protein synthesis system comprising a cell-free extract, a nucleic acid coding for said protein, and amino acids for the substrate of said protein, wherein said amino acids comprises at least one amino acid comprising a heavy atom, and wherein the introduced rate of said amino acid comprising the heavy atom into the synthesized protein is at least 80%.

Methods for producing proteins by using cell-free protein synthesis systems

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Yokohama, Japan

Takashi Yabuki