Production of soluble recombinant protein by pi value control of n-terminal

An expression vector for improving secretion efficiency of a foreign protein containing a gene construct which comprises:

• (i) a promoter;
• (ii) a polynucleotide operably linked to the promoter encoding a polypeptide fragment containing a signal sequence and/or N-region of the leader sequence or variants thereof in which the pI value is controlled;
• and
• (iii) a polynucleotide operably linked to the polynucleotide encoding the polypeptide fragment or variants thereof encoding a secretional enhancer comprising a hydrophilicity enhancing sequence with the controlled pI value.

A method for improving secretion efficiency of a foreign protein comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 3.09-9.89 or 9.90-11.28;
• 2) regulating the distance between amino acids which affect the pI value in the leader sequence;
• 3) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), the controlled distance region of step 2), a protease recognition site and the foreign protein in that order;
• 4) constructing a recombinant expression vector by inserting operably the gene construct of step 3) into a general expression vector;
• 5) generating a transformant by transforming a host cell with the recombinant expression vector of step 4);
• and,
• 6) selecting a transformant from the culture of the transformant of step 5) exhibiting the highest soluble expression of the target protein.

A method for improving secretion efficiency of a foreign protein comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 2.0-4.9 or 5.0-11.0;
• 2) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), a hydrophilic secretional enhancer, a protease recognition site and the foreign protein in that order;
• 3) constructing a recombinant expression vector by inserting the gene construct of step 2) operably into a general expression vector;
• 4) generating a transformant by transforming a host cell with the recombinant expression vector of step 3);
• and
• 5) selecting a transformant from the culture of the transformant of step 4) exhibiting the highest soluble expression of the target protein.

A method for producing a recombinant fusion foreign protein comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 3.09-9.89 or 9.90-11.28;
• 2) regulating the distance between amino acids which affect the pI value in the leader sequence;
• 3) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), the controlled distance region of step 2), a protease recognition site and the foreign protein in that order;
• 4) constructing a recombinant expression vector by inserting operably the gene construct of step 3) into a general expression vector;
• 5) generating a transformant by transforming a host cell with the recombinant expression vector of step 4);
• 6) culturing the transformant of step 5);
• and
• 7) separating the recombinant fusion foreign protein from the culture solution of step 6).

A method for producing a recombinant fusion foreign protein comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 2.0-4.9 or 5.0-11.0;
• 2) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), a hydrophilic secretional enhancer, a protease recognition site and the foreign protein in that order;
• 3) constructing a recombinant expression vector by inserting the gene construct of step 2) operably into a general expression vector;
• 4) generating a transformant by transforming a host cell with the recombinant expression vector of step 3);
• 5) culturing the transformant of step 4);
• and
• 6) separating the recombinant fusion foreign protein from the culture solution of step 5).

A method for producing a foreign protein in native form comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 3.09-9.89 or 9.90-11.28;
• 2) regulating the distance between amino acids which affect the pI value in the leader sequence;
• 3) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), the controlled distance region of step 2), a protease recognition site and the foreign protein in that order;
• 4) constructing a recombinant expression vector by inserting operably the gene construct of step 3) into a general expression vector;
• 5) generating a transformant by transforming a host cell with the recombinant expression vector of step 4);
• 6) culturing the transformant of step 5);
• 7) separating the recombinant fusion foreign protein from the culture solution of step 6);
• and,
• 8) separating the foreign protein in native form after cleaving the fusion foreign protein of step 7) with a protease that could cleave the protease recognition site.

A method for producing a foreign protein in native form comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 2.0-4.9 or 5.0-11.0;
• 2) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), a hydrophilic secretional enhancer, a protease recognition site and the foreign protein in that order;
• 3) constructing a recombinant expression vector by inserting the gene construct of step 2) operably into a general expression vector;
• 4) generating a transformant by transforming a host cell with the recombinant expression vector of step 3);
• 5) culturing the transformant of step 4);
• 6) separating the fusion foreign protein from the culture solution of step 5);
• and,
• 7) separating the foreign protein in native form after cleaving the fusion foreign protein of step 6) with a protease that could cleave the protease recognition site.

A method for producing an intracellular carrier for the delivery of a target material comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 3.09-9.89 or 9.90-11.28;
• 2) regulating the distance between amino acids which affect the pI value in the leader sequence;
• 3) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), the controlled distance region of step 2), a protease recognition site and the foreign protein in that order;
• 4) constructing a recombinant expression vector by inserting operably the gene construct of step 3) into a general expression vector;
• 5) generating a transformant by transforming a host cell with the recombinant expression vector of step 4);
• 6) culturing the transformant of step 5);
• 7) separating the fusion foreign protein from the culture solution of step 6);
• 8) separating the peptide containing the leader sequence, the hydrophilic secretional enhancer and the protease recognition site but not the foreign protein in native form after cleaving the fusion foreign protein of step 7) with a protease that could cleave the protease recognition site;
• and,
• 9) combining the peptide containing the leader sequence, the hydrophilic secretional enhancer and the protease recognition site of step 8) with a target material which is supposed to be delivered into the cell.

A method for producing an intracellular carrier for the delivery of a target material comprising the following steps:

• 1) designing a leader sequence having a signal sequence and/or N-region of the leader sequence of a foreign protein with the controlled pI value of 2.0-4.9 or 5.0-11.0;
• 2) constructing a gene construct composed of a polynucleotide encoding a fusion protein containing the leader sequence of step 1), a hydrophilic secretional enhancer, a protease recognition site and the foreign protein in that order;
• 3) constructing a recombinant expression vector by inserting the gene construct of step 2) operably into a general expression vector;
• 4) generating a transformant by transforming a host cell with the recombinant expression vector of step 3);
• 5) culturing the transformant of step 4);
• 6) separating the fusion foreign protein from the culture solution of step 5);
• 7) separating the peptide containing the leader sequence, the hydrophilic secretional enhancer and the protease recognition site but not the foreign protein in native form after cleaving the fusion foreign protein of step 6) with a protease that could cleave the protease recognition site;
• and
• 8) combining the peptide containing the leader sequence, the hydrophilic secretional enhancer and the protease recognition site of step 7) with a target material which is supposed to be delivered into the cell.