Process for production of proteins as soluble proteins
The object of the invention is to produce a target protein as a soluble protein using a recombinant protein expression system. The invention provides a process for producing a target protein as a soluble protein, comprising the step of expressing a protein by using a polynucleotide containing, in order, a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a basic amino acid-rich polypeptide, and a polynucleotide encoding the target protein; and also provides an expression vector used for this purpose.
1 . A process for producing a target protein as a soluble protein, comprising the step of expressing a protein by using a polynucleotide comprising a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a basic amino acid-rich polypeptide, and a polynucleotide encoding the target protein.
2 . The process of claim 1 , wherein protein expression is carried out in a host cell.
3 . The process of claim 2 , wherein the host cell is a gram-negative bacterium.
4 . The process of claim 3 , wherein the gram-negative bacterium is a bacterium of the genus Escherichia.
5 . The process of claim 1 , wherein the secretory signal peptide is a secretory signal peptide from a gram-negative bacterium.
6 . The process of claim 5 , wherein the secretory signal peptide from a gram-negative bacterium is a secretory signal peptide from a facultative anaerobic bacillus.
7 . The process of claim 5 , wherein the secretory signal peptide from a gram-negative bacterium is a secretory signal peptide from at least on of the outer membrane protein A of Escherichia coli (OmpA) and a secretory signal peptide from cholera toxin from Vibrio cholerae.
8 . The process of claim 1 , wherein the basic amino acid-rich polypeptide is a polypeptide composed of from approximately 5 to approximately 12 amino acid residues.
9 . The process of claim 1 , wherein the basic amino acid-rich polypeptide has a basic amino acid content of at least approximately 60%.
10 . The process of claim 1 , wherein the basic amino acid in the basic amino acid-rich polypeptide is selected from the group of histidine, arginine and lysine.
11 . The process of claim 1 , wherein the basic amino acid-rich polypeptide is polyhistidine.
12 . The process of claim 1 , wherein expression of the target protein is carried out using an expression vector comprising a polynucleotide encoding the target protein.
13 . A process for producing a target protein as a soluble protein, comprising the step of expressing a protein in a gram-negative bacterium by using a polynucleotide comprising a polynucleotide encoding a secretory signal peptide of the gram-negative bacterium, a polynucleotide encoding a polypeptide composed of from approximately 5 to approximately 12 basic amino acid residues, and a polynucleotide encoding the target protein.
14 . A process for producing a target protein as a soluble protein, comprising the step of expressing a protein in a genus Escherichia bacterium by using a polynucleotide comprising a polynucleotide encoding OmpA, a polynucleotide encoding polyhistidine, and a polynucleotide encoding the target protein.
15 . The process of claim 1 , wherein the target protein is a heterologous protein.
16 . The process of claim 1 , wherein the target protein is selected from the group of apoRLBP, apoaequorin, apoclytin, apoobelin and apomitrocomin.
17 . The process of claim 16 , wherein the target protein is apoRLBP.
18 . A process for producing apoRLBP, comprising the step of expressing a protein within a gram-negative bacterium by using a polynucleotide comprising a polynucleotide encoding a secretory signal peptide of the gram-negative bacterium, a polynucleotide encoding a polypeptide composed of from approximately 5 to approximately 12 basic amino acid residues, and a polynucleotide encoding apoRLBP.
19 . A process for producing apoRLBP, comprising the steps of:
expressing a protein within E. coli by using a polynucleotide comprising a polynucleotide encoding OmpA, a polynucleotide encoding polyhistidine, and a polynucleotide encoding apoRLBP; and
accumulating the expressed protein in the periplasmic space of E. coli.
20 . A process for producing RLBP, comprising the step of contacting the apoRLBP produced by the process of claim 17 with coelenterazine or a derivative thereof.
21 . A process for preserving coelenterazine or a derivative thereof, comprising the step of preparing RLBP by contacting the apoRLBP produced by the process of claim 17 with coelenterazine or a derivative thereof.
22 . RLBP comprising apoRLBP produced by the process of claim 17 and coelenterazine or a derivative thereof.
23 . RLBP comprising apoRLBP produced by the process of claim 18 and coelenterazine or a derivative thereof.
24 . RLBP comprising apoRLBP produced by the process of claim 19 and coelenterazine or a derivative thereof.
25 . An expression vector comprising:
(a) a first coding region which encodes a secretory signal peptide;
(b) a second coding region which encodes a basic amino acid-rich polypeptide; and
(c) at least one restriction enzyme site at which can be inserted a third coding region which encodes a target protein.
26 . The expression vector of claim 25 , wherein the secretory signal peptide is a secretory signal peptide from a gram-negative bacterium.
27 . The expression vector of claim 26 , wherein the secretory signal peptide from a gram-negative bacterium is a secretory signal peptide from a facultative anaerobic bacillus.
28 . The expression vector of claim 26 , wherein the secretory signal peptide from a gram-negative bacterium is a secretory signal peptide from at least one of the outer membrane protein A of Escherichia coli (OmpA) and a secretory signal peptide from cholera toxin from Vibrio cholerae.
29 . The expression vector of claim 25 , wherein the basic amino acid-rich polypeptide is a polypeptide composed of from approximately 5 to approximately 12 amino acid residues.
30 . The expression vector of claim 25 , wherein the basic amino acid-rich polypeptide has a basic amino acid content of at least approximately 60%.
31 . The expression vector of claim 25 , wherein the basic amino acid in the basic amino acid-rich polypeptide is selected from the group of histidine, arginine and lysine.
32 . The expression vector of claim 25 , wherein the basic amino acid-rich polypeptide is polyhistidine.
33 . An expression vector comprising:
(a) a first coding region which encodes a secretory signal peptide from a gram-negative bacterium;
(b) a second coding region which encodes a polypeptide consisting of from approximately 5 to approximately 12 basic amino acid residues; and
(c) at least one restriction enzyme site at which can be inserted a third coding region which encodes a target protein.
34 . An expression vector comprising:
(a) a first coding region which encodes OmpA;
(b) a second coding region which encodes polyhistidine; and
(c) at least one restriction enzyme site at which can be inserted a third coding region which encodes a target protein.