Protein fragments for use in protein targeting

Details Protein fragments for use in protein targeting Protein fragments for use in protein targeting

1998-12-01

2002-01-22

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S005000, C530S326000, C530S330000, C530S387300

Reexamination Certificate

active

06340577

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the use of polypeptides derivable from the core protein of the hepatitis C virus for targeting proteins of interest to lipid globules, in particular lipid globules subsequently secreted into animal milk. The resulting protein/lipid complexes may be used in therapy including the production of vaccines.
BACKGROUND TO THE INVENTION
Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and liver disease. It is estimated that, worldwide, approximately 300 million individuals are infected with the virus, 20% of whom are likely to develop mild to severe liver disease or carcinoma. Apart from the risk of succumbing to the long term effects of infection, these individuals also represent a large reservoir of virus for future transmissions. To date, the only widely used therapy for HCV is treatment with interferon. However, sustained response is achieved in only about 20% of cases. Moreover, no vaccine currently exists to protect against infection. Since growth of the virus has not been possible to date in tissue culture systems, very little is known also about the molecular events which occur during viral replication.
The core protein of HCV is predicted to constitute the capsid of virus particles. From various studies, expression of this protein results in a range of effects on intracellular processes, including a decrease in transcription of genes from HBV and HIV and alterations to apoptosis. There is also evidence from a study on transgenic mice that liver-specific expression of core may be linked to the development of steatosis (fatty liver), a condition commonly found in HCV-infected individuals which is characterised by the accumulation of fat deposits within hepatocytes. Thus, core protein may also influence lipid metabolism within the liver. Other results from studies on human sera suggest that HCV virus particles are found associated with lipoprotein particles which are produced by the liver. It has also been shown that HCV core protein associates with lipid droplets within cells (Barba, G. et al., 1997; Moradpour, D. et al., 1996). The droplets are storage compartments for both triacylglycerols and cholesterol esters which can be used as substrates for oxidation in mitochondria and for the formation of membranes. In specialised cells, stored cholesterol is used for steroid hormone synthesis.
Within the liver, lipid droplets also function as a site for storage of precursors of the lipid which is secreted from this organ in the form of lipoprotein particles. Although lipid droplets were identified several decades ago and they can be readily detected by staining methods, very little is known about the processes of assembly, storage and disassembly within the cell. One protein, termed adipocyte-related differentiation protein (ADRP), has been found to associate with lipid droplets in a range of cell types and in certain organs. To date, it is the only protein which is apparently not cell-type specific that has this intracellular distribution. It is proposed that ADRP may be required for maintenance of lipid droplets within cells, however the precise function of the protein has not been identified.
SUMMARY OF THE INVENTION
Particular sequences within the hepatitis C virus core protein that direct association of HCV core protein with intracellular lipid globules have now been characterised. These sequences can thus be used to target other proteins to lipid globules, including lipid globules secreted by milk-producing cells. We have also shown that expression of core protein and its resultant association with lipid droplets results in loss of ADRP from the droplets. Furthermore, progressive increases in core expression result in diminishing amounts of ADRP to undetectable levels. Since it has been shown previously that ADRP is also secreted as a component of fat globules in milk from humans, cows and rats, proteins comprising HCV core protein sequences may also be secreted into animal milk. Thus fusion proteins comprising HCV core protein elements fused to proteins of interest may be targeted specifically to lipid globules secreted into the milk produced by a variety of animals and the proteins extracted from the milk. This will facilitate the expression and secretion into milk of proteins of interest and provide an effective method of producing recombinant proteins in transgenic animals.
Accordingly, the present invention provides a protein comprising a lipid globule targeting sequence linked to a protein of interest (POI) wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or fragment or homologue thereof. Preferably, the lipid globule targeting sequence comprises amino acids from 125 to 144 and/or 161 to 166 of the HCV core protein as set out in SEQ ID. Nos. 2 and 3, or the equivalent amino acids in other HCV strains/isolates. More preferably, the lipid globule targeting sequence also comprises a hydrophilic amino acid sequence of at least 8 amino acids. The present invention also provides an isolated polypeptide consisting essentially of a lipid globule targeting sequence wherein the targeting sequence comprises from amino acids 125 to 144 and 161 to 166 of an HCV core protein linked to a hydrophilic amino acid sequence of at least 8 amino acids.
The protein of interest is preferably a protein expressed by a pathogen, preferably a viral or bacterial protein or fragment thereof, more preferably comprising at least one epitope.
In another aspect, the present invention provides a polynucleotide encoding a protein of the invention. The present invention also provides a polynucleotide encoding a protein of the invention operably linked to a control sequence permitting expression of the protein in a suitable host cell. Preferred host cells include adipocytes and milk-secreting cells.
The invention further provides a nucleic acid vector comprising a polynucleotide of the invention. The invention also provides a host cell comprising a polynucleotide of the invention or a nucleic acid vector of the invention.
In another aspect, the present invention provides a method for producing a protein of the invention which method comprises culturing a host cell of the invention under conditions which allow expression of the protein, and recovering the protein.
The proteins of the invention may advantageously be extracted from cells associated with the lipid globules to which the proteins have been directed by the lipid globule targeting sequence. In particular, proteins produced in milk-secreting cells in milk-producing animals may conveniently be extracted from the animal's milk. These protein/lipid complexes may be used without further purification. Indeed, lipids have been used as adjuvants in the preparation of vaccine compositions. Consequently, protein/lipid globule compositions of the invention may be used in the preparation of vaccines, in particular where the protein of interest is immunogenic.
Thus, the invention also provides a composition comprising a protein of the invention and a lipid globule. Preferably the lipid globule is a consituent of mammalian milk.
The compositions, proteins, polynucleotides and vectors of the present invention may be used in the prevention or treatment of pathogenic infections. Thus, in a further aspect, the present invention provides a vaccine composition comprising a composition, protein, polynucleotide or vector of the invention together with a pharmaceutically acceptable carrier or diluent. It may be preferred to use the proteins of the invention in combination with the active constituents of other vaccine compositions to increase their effectiveness.
The present invention also provides a method of treating or preventing a pathogenic infection in a human or animal which comprises administering to the human or animal an amount of a composition, protein, polynucleotide or vector of the invention sufficient to achieve a beneficial immunological effect.
DETAILED DESCRIPTION OF THE INVENTION
Although in general the techniques mentioned herein are well known i

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