Method for recombinant yeast expression and isolation of water-soluble collagen-type polypeptides

A method for preparing and isolating a recombinant water-soluble collagen polypeptide comprising the steps of:

• A) preparing and expressing a water-soluble collagen polypeptide in a culture of Saccharomyces cerevisiae cells using recombinant DNA technology, said culture comprising from about 10 to about 30 mmol of calcium ions,
• B) after centrifugation of said culture to remove said cells, isolating said expressed water-soluble collagen polypeptide from the supernatant by subjecting the resulting supernatant to isoelectric precipitation at a pH of from about 3 to about 4,
• C) after centrifugation of the supernatant obtained in step B, resuspending the resulting pellet in a buffered solution of a calcium salt,
• C1) after heating said buffered solution to at least about 45.degree. C. for at least 10 minutes, mixing said solution with a protease to form a proteolytic solution having a pH of from about 7 to about 9,
• D) after centrifugation of said solution, subjecting the supernatant thereby obtained to step gradient ion exchange chromatography to capture said water-soluble collagen polypeptide using buffered solutions of sodium chloride and a DEAE chromatography column,
• E) after heating at least one elution fraction obtained in step D to at least about 45.degree. C. for at least 10 minutes, and subsequently cooling, subjecting said heated and cooled elution fraction to hydrophobic interaction chromatography using ammonium sulfate solutions and a phenyl sepharose chromatography column,
• E1) subjecting at least one elution fraction obtained in step E to diafiltration to replace ions in said elution fraction with different ions, and
• F) recovering said water-soluble collagen polypeptide from the resulting diafiltered elution obtained in step E1, and
• F1) vacuum drying said recovered water-soluble collagen polypeptide.

A method for preparing and isolating a recombinant water-soluble collagen polypeptide comprising the steps of:

• A) preparing and expressing a water-soluble collagen polypeptide in a culture of Saccharomyces cerevisiae cells using recombinant DNA technology, said culture comprising from about 10 to about 30 mmol of calcium ions,
• B) after centrifugation or diafiltration of said culture to remove said cells, isolating said expressed water-soluble collagen polypeptide from the supernatant by subjecting the resulting supernatant to isoelectric precipitation at a pH of from about 3 to about 4,
• C) after centrifugation of the supernatant obtained in step B, resuspending the resulting pellet in a buffered salt solution, said solution having a pH of from about 7 to about 9,
• D) after centrifugation of said buffered salt solution, subjecting the supernatant thereby obtained to a first step gradient ion exchange chromatography to capture said water-soluble collagen polypeptide using buffered solutions of sodium chloride and a DEAE chromatography column,
• D1) adding a buffered solution of a calcium salt and a protease to at least one elution fraction obtained in step D to form a proteolytic solution,
• D2) adding a calcium ion chelating agent to said proteolytic solution,
• E) subjecting said proteolytic solution to a second step gradient ion exchange chromatography using sodium chloride solutions and a DMAE chromatography column,
• E1) subjecting at least one elution fraction obtained in step E to diafiltration to replace ions in said elution fraction with different ions,
• F) recovering said water-soluble collagen polypeptide from the resulting diafiltered elution fraction obtained in step E1, and
• F1) vacuum drying said recovered water-soluble collagen polypeptide.

A method for preparing and isolating a recombinant water-soluble collagen polypeptide comprising the steps of:

• A) preparing and expressing a water-soluble collagen polypeptide in a yeast host cell culture containing yeast host cells using recombinant DNA technology, said culture comprising from about 10 to about 30 mmol of calcium ions,
• B) after centrifugation or diafiltration of said culture to remove said yeast host cells, isolating said expressed water-soluble collagen polypeptide from the supernatant by isoelectric precipitation at a pH which is within .+-.1 pH unit of the isoelectric point of said water-soluble collagen polypeptide,
• C) after centrifugation of the supernatant obtained in step B, resuspending the resulting pellet in a buffer,
• D) after centrifugation of the buffered solution formed in step C, subjecting the supernatant thereby obtained to ion exchange chromatography to capture said water-soluble collagen polypeptide,
• E) obtaining at least one elution fraction and subjecting it to either hydrophobic interaction chromatography or a second ion exchange chromatography, and
• F) recovering said water-soluble collagen polypeptide from at least one elution fraction obtained in step E,
• wherein between steps B and E, treating said water-soluble collagen polypeptide, independently, with a calcium salt and a protease,
• wherein said water-soluble collagen polypeptide comprises two or more occurrences of the Gly-Pro-Y triplet, wherein Y is Gln, Glu, Met, Ile, His, Lys, Asn or Tyr.

A method for preparing and isolating a recombinant water-soluble collagen polypeptide comprising the steps of:

• A) preparing and expressing a water-soluble collagen polypeptide in a yeast host cell culture containing yeast host cells using recombinant DNA technology, said culture comprising from about 10 to about 30 mmol of calcium ions,
• B) after centrifugation or diafiltration of said culture to remove said yeast host cells, isolating said expressed water-soluble collagen polypeptide from the supernatant by isoelectric precipitation at a pH which is within .+-.1 pH unit of the isoelectric point of said water-soluble collagen polypeptide,
• C) after centrifugation of the supernatant obtained in step B, resuspending the resulting pellet in a buffer,
• D) after centrifugation of the buffered solution formed in step C, subjecting the supernatant thereby obtained to ion exchange chromatography to capture said water-soluble collagen polypeptide,
• E) obtaining at least one elution fraction and subjecting it to either hydrophobic interaction chromatography or a second ion exchange chromatography, and
• F) recovering said water-soluble collagen polypeptide from at least one elution fraction obtained in step E,
• wherein between steps B and E, treating said water-soluble collagen polypeptide, independently, with a calcium salt and a protease,
• wherein said water-soluble collagen polypeptide comprises one of the following sequences:
• ##STR6## wherein Xaa.sub.1 and Xaa.sub.2 are independently the amino acids identified as Met, Ile, His, Lys, Asn, Tyr or Gln, and n is 1 to 25.