Method for soluble expression and purification of hydrophobin

Abstract

A method of expressing hydrophobin in a soluble form and a method of purifying hydrophobin, and more particularly, to a method of expressing a recombinant fusion protein including hydrophobin in a soluble form in a host cell and then purifying the expressed recombinant fusion protein are provided.

1 . A recombinant fusion protein expressed in a soluble form, comprising:
 a target protein; and   a ramp tag for controlling a translation rate, fused at an N-terminal of the target protein,   wherein a signal sequence of the N-terminal of the target protein is subjected to mutation.   
     
     
         2 . The recombinant fusion protein of  claim 1 , wherein the target protein is hydrophobin. 
     
     
         3 . The recombinant fusion protein of  claim 2 , wherein the target protein is a Class I hydrophobin. 
     
     
         4 . The recombinant fusion protein of  claim 3 , wherein the target protein is DewA. 
     
     
         5 . The recombinant fusion protein of  claim 1 , wherein a wild type target protein consists of an amino acid sequence of SEQ ID NO: 2. 
     
     
         6 . The recombinant fusion protein of  claim 1 , wherein the target protein consists of an amino acid sequence of SEQ ID NO: 8 or 10. 
     
     
         7 . The recombinant fusion protein of  claim 1 , wherein the ramp tag consists of an amino acid sequence of SEQ ID NO: 5. 
     
     
         8 . The recombinant fusion protein of  claim 1 , wherein the signal sequence consists of a base sequence of SEQ ID NO: 3. 
     
     
         9 . The recombinant fusion protein of  claim 1 , wherein the mutation is one or two or more selected from the group consisting of a substitution and a deletion of a part or all of amino acids of the signal sequence and an addition of a new amino acid. 
     
     
         10 . A base sequence encoding the recombinant fusion protein of  claim 1 . 
     
     
         11 . A vector for soluble expression of the recombinant fusion protein, the base sequence of  claim 10  being introduced into the vector. 
     
     
         12 . A transformant for soluble expression of the recombinant fusion protein, the transformant being transformed with the vector of  claim 11 . 
     
     
         13 . A method of purifying a recombinant fusion protein expressed in a soluble form, the method comprising:
 a) expressing a recombinant fusion protein in a soluble form by transforming a non-human host cell with a vector into which a base sequence encoding a recombinant fusion protein is introduced, the recombinant fusion protein including: a target protein; and a ramp tag for controlling a translation speed, fused at an N-terminal of the target protein; and   b) purifying the expressed recombinant fusion protein.   
     
     
         14 . The method of  claim 13 , wherein b) includes:
 b1) suspending the transformed cell and lysing and centrifuging the suspended cell to obtain a first supernatant;   b2) shaking and then centrifuging a first mixture obtained by adding a first solvent to the first supernatant to obtain a second supernatant; and   b3) shaking and then centrifuging a second mixture obtained by adding a second solvent to the second supernatant to recover the target protein.   
     
     
         15 . The method of  claim 13 , wherein the target protein is hydrophobin. 
     
     
         16 . The method of  claim 15 , wherein the target protein is Class I hydrophobin. 
     
     
         17 . The method of  claim 16 , wherein the target protein is DewA. 
     
     
         18 . The method of  claim 13 , wherein a wild type target protein consists of an amino acid sequence of SEQ ID NO: 2. 
     
     
         19 . The method of  claim 13 , wherein the target protein consists of an amino acid sequence of SEQ ID NO: 8 or 10. 
     
     
         20 . The method of  claim 13 , wherein the ramp tag is obtained by collecting a rare codon of the host cell. 
     
     
         21 . The method of  claim 13 , wherein the host cell is a bacterium belonging to  Escherichia  sp.,  Salmonellae  sp.,  Yersinia  sp.,  Shigella  sp.,  Enterobacter  sp.,  Pseudomonas  sp.,  Proteus  sp., or  Klebsiella  sp. 
     
     
         22 . The method of  claim 14 , wherein each of the first solvent and the second solvent is isopropyl alcohol. 
     
     
         23 . The method of  claim 22 , wherein a volume ratio of the isopropyl alcohol to the first supernatant is 1:2 or 1:3. 
     
     
         24 . The method of  claim 20 , wherein the ramp tag consists of an amino acid sequence of SEQ ID NO: 5. 
     
     
         25 . The method of  claim 20 , wherein the rare codon is collected by analyzing a frequency of the codon and a number of isoacceptor tRNA genes. 
     
     
         26 . The method of  claim 25 , wherein the frequency of the codon is 0.1 to 1%. 
     
     
         27 . The method of  claim 24 , wherein the number of isoacceptor tRNA genes is 0 to 2. 
     
     
         28 . The method of  claim 13 , wherein the target protein is a physiologically active protein including hormones and receptors thereof, biological response modifiers and receptors thereof, cytokines and receptors thereof, enzymes, antibodies, and antibody fragments. 
     
     
         29 . A recombinant fusion protein expressed in a soluble form, the recombinant fusion protein being purified by the method of  claim 13 .