Method for producing recombinant trypsin
View Patent ↗The present invention concerns a method for producing recombinant trypsin from porcine pancreas in Pichia pastoris which is soluble and secreted into the culture medium, whereby expression at pH 3.0-4.0 substantially prevents activation of trypsinogen to β-trypsin and autolysis of β-trypsin by α-trypsin into ε-trypsin and from there into inactive peptides.
1. A method for the recombinant production of active trypsin comprising the steps:
a) providing a yeast host cell comprising a recombinant nucleic acid that encodes a trypsinogen protein comprising a signal peptide and a shortened propeptide sequence that consists of no more than 10 amino acids, wherein the propeptide retains the amino acid sequence from position 20 to position 25 of SEQ ID NO:1 bound to the N-terminus of the amino acid sequence of trypsin, and wherein said signal peptide is bound to the N-terminus of said propeptide and mediates secretion of trypsinogen from the host cell,
b) culturing the yeast host cell in a first culture medium under conditions promoting the growth of the host cell;
c) culturing the host cell in a second culture medium having a pH of from 3.0 to 4.0 and inducing the expression of said recombinant nucleic acid whereby the yeast host cell expresses a trypsinogen comprising the shortened propeptide in the second culture medium significantly more than a trypsinogen comprising a complete propeptide;
d) isolating the trypsinogen comprising the propeptide from the second culture medium, and
e) cleaving the propeptide sequence by addition of trypsin and/or by autocatalytic cleavage to form active trypsin.
2. The method of claim 1 , wherein Pichia pastoris or Hansenula polymorpha is used as the host cell.
3. The method of claim 1 , further comprising separating non-cleaved trypsinogen molecules from active trypsin.
4. The method of claim 1 , wherein a recombinant nucleic acid with an optimized codon usage for the host cell is used.
5. The method of claim 1 , wherein the recombinant nucleic acid codes for a porcine trypsinogen.
6. The method of claim 1 , wherein the recombinant nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 22.
7. The method of claim 1 , wherein said first culture medium has a pH of from 5 to 7.
8. The method of claim 1 , wherein the expression of the recombinant trypsinogen protein is controlled by an inducible expression control sequence.
9. The method of claim 1 , wherein the first culture medium has a pH of 5 to 7 until a predetermined optical density is obtained and then the yeast host cell is cultured in the second culture medium having a pH of from 3.0 to 4.0 and the expression of the recombinant nucleic acid is induced.
10. The method of claim 1 , wherein the second culture medium containing the yeast host cells is admixed with a buffer containing calcium ions and subsequently the steps (d) and (e) are carried out.