Method for producing by cell-free protein synthesis system using thioredoxin-fused protein expression vector

#The present invention relates to a method for producing a protein by cell-free protein synthesis system, and more particularly to a method for producing a highly hydrophobic protein such as a membrane protein as a soluble protein by cell-free protein synthesis system.

#Recently, DNA sequences of various organisms including human have been analyzed increasingly and rapidly, and with regard to a large number of genes extracted from this enormous genome sequence information, a research called “Structural genomics” became an important field, which is a systematic and comprehensive analysis of the relationship between structure and function of proteins by determining three dimensional structure of proteins encoded by individual genes. In addition, a high-throughput analytical means applicable to a large scale analysis for promoting such a research is desired.

#In this “Structural genomics”, a most important target as a pharmaceutical development among the proteins for their structural analysis may for example be a membrane protein. While a membrane protein is responsible for important cellular functions such as a response to a stimulation, cellular skeleton and adhesion, material transportation and electron transport, it is difficult to be analyzed biochemically because of an extreme difficulty in its isolation and purification.

#FIG. 1 shows the results of the expression of MT43-NTR in a cell-free protein synthesis system by a batch method.

#FIG. 2 shows the results of the expression of thioredoxin fused MT43-NTR in a cell-free protein synthesis system by a batch method according to the present invention.

#FIG. 3 shows the results of the expression of MBP-T43NTR in a cell-free protein synthesis system by a batch method.

#(Target Protein)

#While a target protein in an inventive method includes but not limited to an E. coli protein, yeast protein or mammalian protein, an insoluble protein containing a highly hydrophobic moiety locally in its molecule may for example be capable of exhibiting a satisfactory result. As used herein, a “protein” means a polypeptide comprising two or more amino acids linked via a peptide bond to each other, and is generally one having several tens or more of constituted amino acids. A “protein” or “polypeptide” includes an amino acid sequence modified by natural process such as a post translational processing after translation or a chemical modification known in the art. The modification may occur at any position in a protein including a peptide backbone, amino acid side chain or amino terminal or carboxyl terminal.

#In a preferred embodiment of the invention, a target protein may be a membrane protein such as a receptor protein, channel protein and transporter protein as well as a transcription factor, antibody, signal transducing protein and the like, any of which is an important protein as a pharmaceutical development. As used herein, a “membrane protein” is an intrinsic membrane protein as well as those which are palmitoylated, geranylated or myristoylated and whose modified moiety is embedded in a membrane lipid, or those which are undergoing an interaction with the membrane lipid or membrane protein. An extremely water insoluble property is exhibited especially by an intrinsic membrane protein having a transmembrane site because of the presence of a hydrophobic amino acid sequence aligned so that integration into the membrane lipid can readily be achieved. Since these membrane proteins are readily aggregated and form insoluble precipitations when they are expressed in heterologous hosts using a recombinant DNA technology, it is difficult to prepare a protein which is biologically active and has a correct three dimensional structure. Nevertheless, many of these membrane proteins play important roles such as intracellular signal transduction and growth regulation, and thus are very important targets in the pharmaceutical field.