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Ferritin fusion proteins for use in vaccines and other applications

A fusion protein and ferritin technology, applied in the field of fusion ferritin, can solve problems such as strengthening

Inactive Publication Date: 2005-08-24
NEW CENTURY PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Therefore, none of the previous literature has investigated the use of ferritin or any use of the N- and C-termini on the outer and inner surfaces of the particle, respectively (e.g. Figure 2A and B), and no one has used this structure to strengthen the properties of these proteins or peptides by fusing with ferritin to link other proteins or peptides, resulting in fusion proteins capable of self-assembly

Method used

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  • Ferritin fusion proteins for use in vaccines and other applications
  • Ferritin fusion proteins for use in vaccines and other applications
  • Ferritin fusion proteins for use in vaccines and other applications

Examples

Experimental program
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Effect test

example 1

[0073] Example 1 Fusion protein expressed in granules:

[0074] Human hemoglobin protein α chain is connected to the C-terminus of human ferritin through a single glycine linker recombinant fusion protein

[0075] Particle abbreviation: (F L.G.Hα).

[0076] Molecular cloning: the recombinant protein fused with human hemoglobin α subunit and human ferritin L chain was expressed in Escherichia coli. The full-length cDNA of the hemoglobin α chain was connected to the C-terminus of the ferritin light chain gene by a linker consisting of a glycine ( image 3 ).

[0077] Protein expression and purification: The coding sequence of ferritin / hemoglobin was verified by DNA sequencing. The recombinant transformed protein was expressed in bacteria BL21(DE3). When the transformed bacteria grew to an OD value of 1.0 (600nm), 1mMIPTG was added to activate the T7 promoter, and the expression was induced at 30°C. After expression, the cells were resuspended in B-PER buffer and sonica...

example 2

[0084] Example 2 Fusion protein expressed in granules:

[0085] The silver-enriched polypeptide is bound to human ferritin L chain C via a linker containing 2 glycines recombinant fusion protein

[0086] Particle abbreviation: (F L .GG.Ag4), AG4 refers to NPSSLFRYLPSD (serial number: 1).

[0087] As a metal-extracting polypeptide linked to ferritin, the particle was shown to be in the correct conformation by the following observations:

[0088] 1) The purified expression product eluted by size exclusion gel chromatography has the same retention effect as natural recombinant ferritin (ferritin molecular weight: 408K).

[0089] 2) Figure 7 and protein light scattering experiments in Table 3 show that individual dispersed proteins have a diameter of about 180 angstroms;

[0090] 3) The particle characteristics of silver condensing peptide have been clarified;

[0091] Meas.#

[0092] Table 3 (F L .GG.Ag4) data sheet. The results were detected using a Protein...

example 3

[0093] Example 3 extragranular fusion protein

[0094] HIV Tat protein (84 amino acids) through a linker containing 6 glycines Linked to the N-terminus to form a recombinant protein.

[0095] Particle abbreviation: (Tat.6G.F L )

[0096] in:

[0097] HIV Tat protein sequence:

[0098] MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT

[0099] KALGISYGRK KRRQRRRAHQ NSQTHQASLS KQPTSQPRGD

[0100] PTGPKE - (serial number: 2)

[0101] Glycine linker sequence:

[0102] GGGGGG (serial number: 3)

[0103] Human ferritin L chain sequence:

[0104] MSSQIRQNYS TDVEAAVNSL VNLYLQASYT YLSLGFYFDR DDVALEGVSH

[0105] FFRELAEEKR EGYERLLKMQ NQRGGRALFQ DIKKPAEDEW GKTPDAMKAA

[0106] MALEKKLNQA LLDLHALGSA RTDPHLCDFL ETHFLDEEVK LIKKMGDHLT

[0107] NLHRLGGPEA GLGEYLFERL TLKHD (Serial Number: 4)

[0108] Molecular cloning: expression of wild-type HIV-1 Tat fused with human ferritin L-chain recombinant protein in Escherichia coli. Using the PCR method, the full-length cDNA of the Tat prot...

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Abstract

An isolated ferritin fusion protein is provided in which ferritin is fused with a protein or peptide capable of being fused to ferritin without interfering with the polymeric self-assembly of the resulting fusion protein, and the protein may be of the endocapsid form when fused at the C terminus or an exocapsid form when fused at the N terminus. These fusion proteins may self-assemble into a variety of useful higher polymeric forms, e.g., capsid or other polymeric aggregate, and they are advantageous in that they are useful in a variety of applications, including human and veterinary vaccines and therapeutics, blood substitutes, image contrast agents, metal chelating agents, gelling agents, protein purification platforms, and therapeutic receptor-binding proteins.

Description

[0001] related application [0002] This patent application has the benefit of US Provisional Patent No. 60,379,145, filed May 10, 2002, which is hereby incorporated by reference in its entirety. field of invention [0003] The present invention generally relates to the fusion of ferritin, especially the fusion of other proteins or peptide fragments at the N-terminus and / or C-terminus inside and outside of ferritin to form a fusion protein capable of self-assembly, and the use of this type of fusion protein as a vaccine and other aspects Applications include the transport of oxygen and the therapeutic introduction of drugs and other therapeutic agents. Background of the invention [0004] Ferritin contains 24 highly conserved subunits, which are present in all animals, bacteria and plants. The main physiological role of ferritin is to control polynuclear Fe(III) 2 o 3 Speed ​​and positioning of formation. (See Theil, E.C. et al. in Adv.Enzymol.Relat.Areas Mol.Biol., 1990...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K39/00A61K39/21A61K39/385C07K14/16C07K14/47C07K14/805C12N15/62
CPCC12N2740/16222C07K14/805C07K2319/00C12N2740/16234C07K14/47C07K2319/735A61K39/21C12N2740/16322C12N15/62C07K14/005C07K2319/40A61K39/385C07K2319/10A61K2039/6031A61K38/00C12N2740/16334A61K39/00C12N2770/20022A61K39/12
Inventor 丹尼尔·C·卡特切斯特·Q·李
Owner NEW CENTURY PHARMA INC
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