Production of Biologically Active Proteins

Abstract

A fusion protein that is expressed in a recombinant protein body-like assembly (RPBLA) in host eukaryotic cells and organisms is disclosed. More particularly, a biologically active polypeptide fused to a protein sequence that mediates the induction of RPBLA formation is expressed and accumulated in host cells after transformation with an appropriate vector. The eukaryotic host cell does not produce protein bodies in the absence of the fusion protein. Methods for preparing and using the RPBLAs and the fusion protein are also disclosed, as are nucleic acid molecules that encode the fusion proteins.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims benefit of provisional application Ser. No. 60 / 776,391 that was filed on Feb. 23, 2006.TECHNICAL FIELD[0002]The present invention contemplates the production of biologically active recombinant peptides and proteins, collectively referred to as polypeptides, in eukaryotic cells and organisms as host systems. More particularly, a biologically active polypeptide is fused to a protein body-inducing sequence (PBIS) that mediates the induction of recombinant protein body-like assemblies (RPBLA) to form a fusion protein that is stably expressed and accumulated in the host system as an RPBLA after transformation of the host cells with an appropriate vector.BACKGROUND ART[0003]The production of recombinant proteins for therapeutic, nutraceutical or industrial uses has enjoyed great success over the past several decades. Introduction of heterologous genes having desired nucleotide sequences into a variety of expression hosts ...

AI Technical Summary

Benefits of technology

"The present invention provides a system and method for producing a fusion protein containing a protein body-inducing sequence (PBIS) and a biologically active polypeptide or protein of interest (target) in eukaryotic cells. The PBISs are able to induce the formation of recombinant fusion proteins that accumulate as recombinant protein body-like assemblies (RPBLAs) in the host cells. The RPBLAs are stable and can be isolated and purified from the host cells. The method involves the transformation of host cells with a nucleic acid sequence coding for the fusion protein and a PBIS. The biologically active polypeptide of the fusion protein exhibits at least 25% of the biological activity of the same polypeptide isolated from the second cell type in an assay of the activity of that polypeptide. The nucleic acid sequence used for transformation can also contain a linker or spacer amino acid sequence between the PBIS and the target polypeptide. The invention provides a novel method for producing a biologically active fusion protein in eukaryotic cells."

Problems solved by technology

In many instances, however, the protein or polypeptide that is ultimately produced can possess the correct primary amino acid residue sequence of the naturally-produced molecule, but lack the biological activity expected of that material.

For example, disulfide bond formation occurs spontaneously in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, but not in the reducing environment of the cytosol of prokaryotes, which makes bacterial cells such as Escherichia coli poor hosts for the synthesis of correctly-folded mammalian proteins that are normally stabilized by disulfide bonds.

However, even in this compartment the bacterial oxi-redox system is not very efficient for eukaryotic proteins.

Unfortunately, many such eukaryotic expression systems are inefficient with respect to protein product yield and cost of manufacture, even when proteins are secreted extracellularly.

The high costs frequently derived from low recombinant protein production levels and / or from complicated downstream protein isolation and purification procedures can invalidate a protein's commercial application.

This is an important issue because the efficiency of protein renaturation is highly limiting, particularly if the protein contains disulfide bonds (Clarc, Ed., April 2001 Curr. Opin. Biotechnol. 12(2):202-207).

As a consequence the yield of correctly refolded recombinant proteins from inclusion bodies is extremely low, and moreover the biological activities of such refolded proteins are typically much less than that of the native-formed proteins.

In addition, those authors constructed zein-GFP fusion proteins to determine the subcellular localization of zein proteins in yeast cells but did not observe formation of dense, concentrated structures characteristic of bona fide PBs.

(2002, Plant Cell 14: 655-672) concluded that yeast is a poor model for the study of zein interactions because zeins accumulated very poorly in transformed yeast.

Moreover, the presence of disulfide bonds in some natural PB-assembling protein domains, as for instance RX3, [Ludevid et al., 1984 Plant Mol. Biol. 3:227-234 and Kawagoe et al., 2005 Plant Cell April 17(4):1141-1153], which are probably involved in PB formation and stabilization, could represent an additional difficulty for production of a biologically active, native-folded protein in PBs.

However, the absence of immunomodulatory components having adjuvant properties associated with attenuated or killed vaccines often results in weaker immunogenicity for such vaccines.

Yet, this destructive environment generates peptides that are capable of binding to MHC class II molecules.

To a large degree, this diversity limits the use of molecular targeting techniques to cell surface markers and immune modulators such as cytokines, because for many species including wildlife, only minimal knowledge of these molecules is available.

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