Novel modified nucleic acid sequences and methods for increasing mRNA levels and protein expression in cell systems

Abstract

The invention provides modified recombinant nucleic acid sequences (preferably DNA) and methods for increasing the mRNA levels and protein expression of proteins which are known to be, or are likely to be, difficult to express in cell culture systems, mammalian cell culture systems, or in transgenic animals. The preferred “difficult” protein candidates for expression using the recombinant techniques of the invention are those proteins derived from heterologous cells preferably those of lower organisms such as parasites, bacteria, and virus, having DNA coding sequences comprising high overall AT content or AT rich regions and / or mRNA instability motifs and / or rare codons relative to the recombinant expression system to be used.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The invention relates to heterologous gene expression. More particularly, the invention relates to the expression of microbial or parasitic organism genes in higher eukaryote cell systems. [0003] 2. Summary of the Related Art [0004] Recombinant production of certain heterologous gene products is often difficult in in vitro cell culture systems or in vivo recombinant production systems. For example, many researchers have found it difficult to express proteins derived from bacteria, parasites and virus in cell culture systems different from the cell from which the protein was originally derived, and particularly in mammalian cell culture systems. One example of a therapeutically important protein which has been difficult to produce by mammalian cells is the malaria merozoite surface protein (MSP-1). [0005] Malaria is a serious heath problem in tropical countries. Resistance to existing drugs is fast developing and a v...

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Benefits of technology

[0008] The present invention provides improved recombinant DNA compositions and procedures for increasing the mRNA levels and protein expression of proteins derived from heterologous cells, preferably those of lower organisms such as bacteria, virus, and parasite, which have previously been difficult to express in cell culture systems, mammalian cell culture systems, or in transgenic mammals. The preferred protein candidates for expression in an expression system in accordance with the invention are those proteins having DNA coding sequences comprising high overall AT content or AT rich regions, and / or mRNA instability motifs and / or rare codons relative to the recombinant expression systems.

Problems solved by technology

Recombinant production of certain heterologous gene products is often difficult in in vitro cell culture systems or in vivo recombinant production systems.

For example, many researchers have found it difficult to express proteins derived from bacteria, parasites and virus in cell culture systems different from the cell from which the protein was originally derived, and particularly in mammalian cell culture systems.

Malaria is a serious heath problem in tropical countries.

However, a major problem in developing MSP-1 as a vaccine is the difficulty in obtaining recombinant proteins in bacterial or yeast expression systems that are equivalent in immunological potency to the affinity purified native protein (Chang et al., (1992) J. Immunol. 148:548-555.) and in large enough quantities to make vaccine production feasible.

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