Sequence and method for genetic engineering of proteins with cell membrane translocating activity

• US 6,432,680 B1
• Filed: 05/01/2000
• Issued: 08/13/2002
• Est. Priority Date: 03/31/1998
• Status: Expired due to Fees

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Lin, Yao-Zhong, Rojas, Mauricio, Donahue, John P., Tan, ZhongJia
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/562,868
• Time in Patent Office: 834 Days
• Field of Search: 435/69.7, 435/193, 435/252.3, 435/320.1, 514/2, 514/12, 530/300, 530/350
• US Class Current: 435/69.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 9/1088 Glutathione transferase (2.5.1.18)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention describes a membrane-translocating peptide sequence (MTS) which facilitates entry of polypeptides and proteins into cells. Also described is an isolated nucleotide sequence encoding the membrane-translocating peptide and a method of using this sequence to genetically engineer proteins with cell membrane permeability. The MTS, and the method of genetically engineering proteins with cell membrane permeability, are useful for polypeptide and protein delivery for human and veterinary applications such as vaccine delivery and cancer therapy.

Claims

1. An isolated fusion polypeptide comprising:

a) a membrane-translocating peptide of about 8 to about 50 residues comprising at least eight consecutive residues of SEQ ID NO;

1 (Ala-Ala-Val-Leu-Leu-Pro-Val-Leu-Leu-Ala-Ala-Pro), and

b) a target peptide or a target polypeptide.

2. The isolated fusion polypeptide of claim 1, wherein the membrane-translocating peptide comprises at least nine consecutive residues of SEQ ID NO:

1.

3. The isolated fusion polypeptide of claim 1, wherein the membrane-translocating peptide comprises at least ten consecutive residues of SEQ ID NO:

1.

4. The isolated fusion polypeptide of claim 1, wherein the membrane-translocating peptide comprises at least eleven consecutive residues of SEQ ID NO:

1.

5. The isolated fusion polypeptide of claim 1, wherein the membrane-translocating peptide comprises at least twelve consecutive residues of SEQ ID NO:

1.

6. The isolated fusion polypeptide of claim 1, wherein the fusion polypeptide comprises a target peptide.

7. The isolated fusion polypeptide of claim 1, wherein the fusion polypeptide comprises a target polypeptide.

8. The isolated fusion polypeptide of claim 1, wherein the target polypeptide comprises a protein domain.

9. The isolated fusion polypeptide of claim 1, wherein the target polypeptide comprises a protein.

10. The isolated fusion polypeptide of claim 1, wherein the target polypeptide comprises a complex formed by two or more polypeptides.

11. The isolated fusion polypeptide of claim 1, wherein the target polypeptide comprises a cell cycle regulatory protein.

12. The isolated fusion polypeptide of claim 1, wherein the target polypeptide comprises an intracellular enzyme.