Functional recombinantly prepared synthetic protein polymer

• US 5,514,581 A
• Filed: 11/06/1990
• Issued: 05/07/1996
• Est. Priority Date: 11/04/1986
• Status: Expired due to Term

• Current Assignee: Protein Polymer Technologies, Inc., Spine Wave Incorporated
• Original Assignee: Protein Polymer Technologies, Inc.
• Inventors: Ferrari, Franco A., Cappello, Joseph
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US07/609,716
• Time in Patent Office: 2,009 Days
• Field of Search: 530/330, 435/172.3, 435/69.1, 435/320.1, 435/252.3, 435/252.33, 576/27, 536/23.5, 536/23.4
• US Class Current: 435/252.3
• CPC Class Codes: A61L 17/00 Materials for surgical sutures or for ligaturing blood vessels surgical adhesives A61L24/00; surgical instruments, devices or methods for suturing or ligaturing A61B17/04, A61B17/12; supports or packages for suture materials A61B17/04; Materials for prostheses or catheters bone cements or surgical adhesives for soft body tissues A61L24/00; shape or structure of prostheses A61F2/00; shape or structure of catheters A61M5/00
• CPC Class Codes: A61L 29/049 Mixtures of macromolecular compounds
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/43586 from silkworms
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 17/08 the carrier being a synthetic polymer
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C08L 89/00 Compositions of proteins; Compositions of derivatives thereof foodstuff preparations A23J3/00
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: G03C 1/047 : Proteins, e.g. gelatine derivatives; Hydrolysis or extraction products of proteins

Abstract

Novel polymers are provided which are produced by recombinant techniques. The polymers are characterized by having a small repeating sequence which provides for strands capable of associating, resulting in useful structural characteristics, where the strands are joined by turns or loops which are flexible and available for interaction with the environment. Specifically, repeating groups of naturally occurring proteins such as silk are modified by introduction of an amino-acid sequence at a site which provides for a turn between strands to provide for readily available oligopeptides capable of interacting with molecules in the environment.

Claims

1. A DNA sequence encoding a proteinaceous polymer comprising segments of repeating units of from 3-9 amino acids naturally occurring fibroin, said segments capable of assembling into aligned structures to be formable into articles, said proteinaceous polymer having a molecular weight of at least about 15 Kdal, with at least two segments joined by an intervening oligopeptide other than said repeating units, wherein said intervening oligopeptide is unaligned, said polymer has individual segments of the same or different repeating units, wherein said intervening oligopeptide comprises RGD.

2. A DNA sequence according to claim 1, wherein said segments are of from about 25 to 150 amino acids, and said intervening oligopeptide is between each of said segments and is of from about 4 to 50 amino acids.

3. A DNA sequence according to claim 1, wherein said repeating units comprise the sequence GAGAGS (SEQ ID NO:

36, positions 91-96).

4. A DNA sequence according to claim 1, wherein said intervening oligopeptide includes the amino acid sequence AVTGRGDSPAS (SEQ ID NO:

15).

5. A vector comprising a replication system and a DNA sequence according to claim 1.

6. A vector according to claim 5, wherein said replication system is a prokaryotic system.

7. A prokaryotic cell comprising a vector comprising a replication system and a DNA sequence according to claim 1.

8. A DNA sequence encoding a proteinaceous polymer having a silk protein repeating unit, wherein the polymer is of at least 15 kDal, and is comprised of segments of the formula (G A G A G S)₉ D A A V T G R G D S P A S A A G Y (SEQ ID NO:

36, positions 91-160).