Method and composition for a protein transduction technology and its applications

• US 8,722,348 B2
• Filed: 05/28/2008
• Issued: 05/13/2014
• Est. Priority Date: 05/28/2008
• Status: Active Grant

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Wang, Jianjun, Li, Qianqian
• Primary Examiner(s): Shen, Bin
• Application Number:
  US12/128,320
• Publication Number:
  US 20090298111A1
• Time in Patent Office: 2,176 Days
• Field of Search: 435/7.21
• US Class Current: 435/7.21
• 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: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

A protein transduction method for efficiently delivery of exogenous proteins into mammalian cells is invented, which has the capability of targeting different cellular compartments and protection from degradation of the delivered proteins from cellular proteases. A composition for treat proteins has cation reagents, lipids and enhancers in a carrier. The method can be used in a number of ways including: production of large quantities of properly folded, post-translationally modified proteins using mammalian cell machinery, a in-cell fluorescence spectroscopy and imaging using small molecule fluorophores and a in-cell NMR spectroscopy using living mammalian cells. The method permits cell biology at atomic resolution that is physiologically and pathological relevant and permits protein therapy to treat human diseases. The method can also be used to deliver exogenous protein inside mammalian cells, wherein the exogenous proteins follow a similar secretion pathway as that of the endogenous protein.

Claims

1. A method of protein transduction into mammalian cells, the method comprising the steps of:

providing a protein of interest;

modifying the protein with protein transduction reagents that enable the protein to be efficiently delivered into mammalian cells, comprising the step of non-covalently binding the reagent to the protein; and

delivering the non-covalently bound protein into mammalian cells,

wherein the protein transduction reagents comprise a cation reagent and a lipid.

2. The method according to claim 1, further including the steps of labeling the protein of interest before delivery into the mammalian cells and monitoring protein activity, folding, post-translational modification, and/or intracellular trafficking and secretion using labeled protein.

3. The method according to claim 1 or 2, further including an in vivo protein refolding method comprising preparing modified proteins of interest, delivering the modified proteins into the correct intracellular compartment of mammalian cells whose intracellular folding machinery refolds the delivered proteins, and purifying properly refolded proteins from cell lysates.

4. The method according to claim 1, wherein said non-covalently binding step including protecting the protein from proteases.

5. The method of claim 1, wherein the protein transduction reagents further comprise an enhancer.

6. The method of claim 1 or claim 5, wherein the cation reagent comprises polyethylenimine.