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Chaperone expression genomes

a technology of genomes and chaperones, applied in the field of chaperone expression genomes, can solve the problem of inability to control the expression ratio of foreign proteins in individual cells, and achieve the effects of facilitating co-translocation, and improving the folding of a protein of interes

Inactive Publication Date: 2008-03-06
BELYAEV ALEXANDER S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a genome that expresses two or more molecular chaperones simultaneously with a protein of interest. This is advantageous because chaperones act in ensemble to assure better folding of a protein of interest. The invention also provides a method for producing inclusion bodies consisting of a protein of interest and large amount of molecular chaperones, which manifests in improved recovery of a biologically active protein. The invention is as convenient and reliable as conventional systems and provides sets of molecular chaperones which improve protein solubility in cases of insoluble proteins. The invention is of general importance for constructing convenient vectors supplemented with molecular chaperones in bacterial or eucaryotic cells.

Problems solved by technology

However, it is difficult, and typically impossible to control the ratio of expression of foreign proteins in individual cells if they are provided on different genomes, for example on different viruses at co-infection, or on different plasmids at co-transfection.

Method used

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Examples

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example 1

Construction of an EGT Transfer Vector (AB-EGT)

[0089] To construct EGT transfer vector according to present invention, a DNA fragment comprising EGT gene polynucleotide sequence is cloned into the pUC19 plasmid vector (Acc# X02514) cleaved with HindIII and SmaI restriction endonucleases (FIG. 2). The EGT gene can be obtained from AcNMPV DNA (Acc# NC—001623, Smith G. E. and Summers, M. D., U.S. Pat. No. 4,879,236, 1987) or any of its derivatives, for example BacPAK6 recombinant baculovirus vector DNA (Kitts P. et al., Biotechniques, 14: 810-817, 1993) or Bac-to-Bac, Bac-N-Blue, BaculoDirect vector DNA (Invitrogen, Carlsbad, Calif.). BacPAK6 DNA is available from several sources, for example Pharmingen (San Diego, Calif.), Clontech (Palo Alto, Calif.), Orbigen, (San Diego, Calif.). Alternatively, any other baculovirus DNA containing EGT gene can be used for this purpose. Primers, complementary to opposite strands of the baculovirus DNA are selected in order to provide for amplificati...

example 2

Multiple EGT Transfer Vector (EGT3)

[0091] Multiple expression vectors are desirable when there is a need to facilitate expression of several genes in the same vector. This example describes insertion of 3 promoters into AB-EGT vector in order to facilitate expression of up to 3 polynucleotide sequences of interest. Expression of a polynucleotide sequences of interest can be achieved using EGT promoter resident in the EGT vectors. However, employment of much stronger promoters, such as polyhedrin, p10 or a basic protein promoter is preferred (Bonning B. C. et al., J. Gen. Virol., 75 (Pt 7):1551-1556, 1994). If the same promoter is employed twice in the same cassette, it is preferred that its copies are facing in opposite directions, more preferably away from each other. This is done in order to avoid unstable direct polynucleotide sequence repeats and to minimize interference between transcription complexes moving along the DNA molecule.

[0092] A promoter cassette containing 3 promo...

example 3

EGT Transfer Vector with a Reporter Gene (EGT3-GFP)

[0095] This example describes insertion of a polynucleotide sequence of interest, which facilitates selection of recombinant baculoviruses obtained by recombination with a backbone plasmid transfer vector. More specifically, it describes insertion of such polynucleotide sequence into the EGT3 transfer vector, and more specifically, insertion of a gene encoding for a reporter protein, such as GFP (FIG. 3). A GFP gene, derived from jellyfish Aequeorea victoria, is amplified in PCR from a DNA containing such gene. Plasmid DNA containing this gene is available from Columbia University, New York or it can be purchased from commercial sources, for instance pGFP plasmid available from (Clontech, Palo Alto, Calif.). DNA containing GFP gene is amplified in PCR using following primers:

[SEQ ID NO: 5]5′-GCGCGGATCCAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGG-3′[SEQ ID NO: 6]5′-GCGCGGATCCTCTTTGTATAGTTCATCCATGCCATGTG-3′

[0096] BamHI restriction endonuclea...

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Abstract

A recombinant genome comprising polynucleotides encoding at least two additional molecular chaperones and a protein of interest, recombinant baculovirus vectors providing molecular chaperones and a method for producing a foreign protein using said genomes and vectors.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of U.S. application Ser. No. 10 / 898,717 filed Jul. 23, 2004, now issued as U.S. Pat. No. 7,226,781; which claims the benefit under 35 USC § 119(e) to U.S. Application Ser. No. 60 / 490,350 filed Jul. 24, 2003, now abandoned. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates in general to a genome expressing additional molecular chaperones simultaneously with a protein of interest in order to assure better folding of a protein of interest. [0004] 2. Background Information [0005] High level expression of proteins, such as for use as biopharmaceuticals, requires proper protein folding for optimal biological activity. Overexpressed proteins are often misfolded and form biologically inactive insoluble protein aggregat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/04C12N15/00
CPCC07K14/005C07K14/47C12P21/02C12N15/86C12N2710/14043C07K2319/00
Inventor BELYAEV, ALEXANDER S.
Owner BELYAEV ALEXANDER S
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