Purification of structurally ordered recombinant protein polymers

• US 5,235,041 A
• Filed: 04/13/1992
• Issued: 08/10/1993
• Est. Priority Date: 12/28/1990
• Status: Expired due to Term

• Current Assignee: Protein Polymer Technologies, Inc.
• Original Assignee: Protein Polymer Technologies, Inc.
• Inventors: Textor, Garret, Buerkle, Tina L., Ferrari, Franco A., Cappello, Joseph
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US07/869,212
• Time in Patent Office: 484 Days
• Field of Search: 530/353, 530/354, 530/355, 530/356, 530/357, 530/370, 530/412, 530/418, 530/420, 530/427, 530/422, 530/423
• US Class Current: 530/353
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Abstract

Recombinant structural and functional polymers are purified by lysing of the cellular host, separation of the solid materials, washing and extraction of contaminants using a detergent solution at elevated temperatures.

Claims

1. A method of purification of substantially insoluble protein polymers, said polymers being characterized by being prepared by recombinant techniques, having long stretches of repeating units of silk fibroin and sufficient stretches of the repeating units so that the polymers have the structural order of silk, said method comprising:

treating and lysing cells to provide lysate comprising said protein polymers in insoluble form;

suspending the disrupted cells from said lysate in an aqueous medium;

separating high molecular weight insoluble material from the suspension;

extracting said separated high molecular weight insoluble material at a temperature in the range of about 60°

to 100°

C. with an aqueous detergent solution; and

isolating the purified protein polymer.

2. A method according to claim 1, wherein lysing comprises mechanical disruption and said separating comprises centrifugation.

3. A method according to claim 1, wherein said extracting is performed at a temperature of at least about 80°

C. and said detergent solution is sodium dodecylsulfate.

4. A method according to claim 1, wherein said isolating is by centrifugation.

5. A method of purification of substantially insoluble protein polymers, said polymers being characterized by being prepared by recombinant techniques, having long stretches of silk fibroin repeating units and having sufficient stretches of the repeating units so that the polymers have the structural order of silk, said method comprising:

treating and lysing cells comprising said protein polymers by mechanical disruption to provide lysate comprising said protein polymers in insoluble form;

separating high molecular weight insoluble material from said lysate by means of centrifugation;

extracting said separated high molecular weight insoluble material at a temperature in the range of about 80°

to 100°

C. with an aqueous anionic sulfuric acid based detergent solution, said detergent being present in from about 1 to 3% w/v; and

isolating the purified protein polymer by centrifugation.

6. A method according to claim 5, wherein said aqueous anionic sulfuric acid based detergent solution is 1.5 to 3% w/v.