Method for enhancing expression of recombinant protein

Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. We offer a variety of recombinant protein expression platforms along with a host of other protein services like chemical protein synthesis, protein refolding and structural biology services.Email:marketing@medicilon.com.cn web:www.medicilon.com

The present invention provides methods for enhancing protein expression from an RNA viral vector and RNA viral vectors with enhanced protein expression capacity. The present inventors successfully increased the level of protein expression significantly by expressing the proteins fused with an AB5B protein from RNA viral vectors. Effective gene therapy, gene vaccination, monoclonal antibody preparation, or such can be achieved by using a gene transfer RNA viral vector of the present invention.

The present invention relates to methods for enhancing the expression of a recombinant protein from an RNA viral vector, methods for preparing recombinant proteins useful for pharmaceuticals and the like by using the above-described method, RNA viral vectors that express the recombinant proteins, and such.

Many recombinant proteins and peptides are currently used as pharmaceutical compositions. It is thought that the type and number of biopharmaceuticals will increase in the future, and that they will form a larger part of pharmaceuticals. Many bioactive proteins and peptides have been suggested as candidates for pharmaceutical compositions. In such cases, the availability and applicability of proteins and peptides can be greatly improved when their preparation is simplified by increasing their expression levels.

The same is also applicable to gene transfer RNA viral vectors used in gene therapy or such. When a gene transfer RNA viral vector is used to apply a gene to gene therapy, genetic vaccine, monoclonal antibody preparation or the like, the expression level of the inserted gene is directly linked to the vector function. Thus, the vector performance can be improved by increasing the expression level, which leads to great improvement of the availability and applicability of proteins.

The AB5 toxin produced by bacteria is a proteinaceous exotoxin produced by toxin-producing bacteria and secreted to the outside of bacterial cells. The AB5 toxin consists of one active subunit (A subunit) having toxicity and five binding subunits (B subunits) having cell-binding activity. Cholera toxin is an AB5 toxin, it has been reported that the nontoxic cholera toxin B subunit (CTB) is applied to infection and chronic diseases as a molecular adjuvant, for example, for influenza virus vaccine , pertussis vaccine , respiratory syncytial (RS) virus vaccine, cancer vaccine , and Alzheimer's disease vaccine . However, there is no report that describes expressing a recombinant protein by using an RNA viral vector or enhancing the expression of a recombinant protein by using an RNA viral vector.

The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide methods for enhancing the expression of recombinant proteins and peptides that are useful as pharmaceutical compositions from RNA viral vectors to simplify the preparation of recombinant proteins and peptides, and provide more effective gene transfer vectors that yield high expression of therapeutic genes by introducing the above-described technology into RNA virus-derived gene transfer vectors when applied in gene therapy, gene vaccination, monoclonal antibody preparation, and such.

The present inventors revealed that when a protein or peptide is expressed from RNA virus, there are not a few cases where the protein or peptide expression level is low and its preparation is difficult due to the property of the protein or peptide to be expressed. In particular, the present inventors found that the expression efficiency is extremely low when short peptides, proteins with low solubility, or such are expressed from RNA viral vectors.

Meanwhile, the AB toxin B subunit (AB5B) is a protein of more than ten KD. Its three-dimensional structural analysis has demonstrated that it forms a pentamer. Cholera toxin B (CTB), which is an AB5B, has been used as an adjuvant. However, it is not known whether the level of protein expression is altered when using CTB. During the course of expressing a recombinant protein as a CTB fusion protein, the present inventors found that the expression level was significantly increased when the recombinant protein was expressed as a CTB fusion protein using a Sendai virus vector as compared to when expressing the recombinant protein alone.

To analyze this phenomenon more closely, the present inventors selected various types of recombinant proteins as an example, and assessed the change (increase or decrease) in their expression levels by fusing them with AB5B. Furthermore, to evaluate the difference between AB5B and other carrier proteins (fusion proteins), the receptor-binding domain (Ia) of Pseudomonas exotoxin A (PEDI) and interleukin-4 (IL-4) were selected and their fusion with AB5B was compared.