Codon optimized sequence for an antiviral protein

Abstract

A codon optimized nucleic acid sequence for Interferon Alpha-2a is provided which can be used for expression of Interferon Alpha-2a in E. Coli.

Claims

1. A recombinant nucleic acid molecule comprising the sequence of SEQ ID NO: 1 encoding Human Interferon alpha-2a, or comprising the sequence which is complementary of SEQ ID NO: 1.

2. An expression vector comprising the nucleic acid molecule of claim 1.

3. The expression vector of claim 2 that is a plasmid or a bacterial phage.

4. An Escherichia coli host cell comprising the expression vector of claim 2.

5. A method of producing recombinant Human Interferon alpha-2a comprising culturing an Escherichia coli host cell comprising an expression vector wherein the expression vector comprises a recombinant nucleic acid molecule with the sequence of SEQ ID NO:1 and isolating Human Interferon alpha-2 produced by the host cell.

6. A recombinant nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 (Sequence ID no 1) illustrates the codon optimized DNA sequence of the optimized Interferon alpha-2a gene.

(2) FIG. 2 illustrates the plasmid utilized to express the codon optimized DNA sequence of Interferon alpha-2a in E. coli.

(3) FIG. 3 illustrates the arrangement of the Codon optimized cDNA gene in the plasmid 1. The upstream promoters and other elements of the vector are illustrated.

(4) FIG. 4 illustrates a comparison of the Native cDNA of Interferon alpha-2a sequence with the codon optimized sequence of Sequence ID no 1.

(5) FIGS. 5A and 5B illustrates a comparison of the yields of protein achieved using the codon optimized and Non optimized cDNA sequence.

DETAILED DESCRIPTION

(6) Accordingly the invention provides a polynucleotide of Interferon alpha-2a (Seq ID no 1) (FIG. 1) that provides significantly higher yield of expression of Interferon alpha-2a using E. Coli as host. The yield of Interferon alpha-2a achieved using optimized interferon alpha-2a cDNA is more as compared to the yield achieved using non optimized cDNA of Interferon alpha-2a. Interferon Alpha: The term interferon-Alpha or IFN-Alpha as used herein refers to Human IFN-Alpha present in the human body and secreted by the leucocytes of the body. The protein is coded by INFA2a gene in humans. Interferon-Alpha can be isolated from natural sources and/or produced by recombinant DNA technology. The said Interferon alpha shall have same sequence homology with, secondary and tertiary structure, bioavailability, potency and the functionality to qualify as a therapeutic biosimilar drug, including bioactivity, of native IFN-Alpha.

(7) Many variants of IFN-Alpha are known in the art. Some mutants are described in details in U.S. patent application Ser. Nos. 11/554,377, 12/542,561, 10/820,467, 10/411,049 incorporated herein by reference herein.

(8) Many methods of cloning and expression of Interferon alpha gene in various hosts like E. coli, Yeast, Animal cells are known in the art. The preferred animal cells are the cell capable of rapidly growth and producing the protein at high expression in continuous cultures, like Chinese hamster ovary cell (CHO).

(9) Expression vectors used for expression of the protein are plasmid, bacterial phage, animal or plant virus, other elements capable of replicating in the host or get integrated in the genome of the host.

(10) Numerous methods are described in the art for expression and purification of Interferon alpha-2a. Some of them are included here by way of reference. U.S. Pat. Nos. 5,196,323, 5,710,027, 7,052,867 Describes method for expression and purification of Interferon alpha-2a in E. coli. U.S. Pat. Nos. 4,680,260, 6,284,520, 7,892,825 describes Process of production of interferon alpha-2a in yeast. U.S. Pat. Nos. 6,159,712, 6,489,144, 4,680,261, 4,966,843 describes method of production of Interferon alpha-2 in mammalian cells.

(11) Codon optimized nucleic acids: Frequency of occurrence of synonymous codons in coding DNA is significantly different in prokaryotic and eukaryotic hosts. This gives rise to significant differences in the composition of their respective genomic tRNA pool in the cytoplasm. When Eukaryotic sequences are cloned into prokaryotic host this factor affects the level of expression of the protein. If the gene insert contains rare codons (codons for which the concentration of the tRNA is less) this can cause a translational pause which can result into detachment of the mRNA from the ribosome. Therefore codon optimization is needed to achieve optimum Expression of the protein in foreign host.

(12) Production of therapeutic proteins using Host Systems like E. coli is carried out to meet the ever increasing demand of therapeutic proteins. Therapeutic proteins produced using such system are costly due to high cost involved in the production of protein. The increase in the level of expression of protein results in production of higher amount of protein per batch thereby reducing the cost of the protein significantly.

EXAMPLE

(13) The following examples are provided to describe the invention and are not intended for reducing the otherwise broad scope of the invention.

Example 1: Codon Optimized cDNA and Vector Containing the Same

(14) Synthetic sequences were synthesized for each individual candidate developed after codon optimization. Such synthetic sequences were cloned into the vector and the said vector was transformed into E. coli (FIG. 2). The expression was analyzed using SDS-PAGE, 2-D gel electrophoresis and other techniques known to the person skilled in the art. The analysis was done for detection of related proteins, no of bands, isoforms of the protein and other properties related to the sequence of the gene. The protein produced was also analyzed as per the test specified in the official monograph of Indian pharmacopeia. Further optimization of the sequence was carried out by identifying the regions on the sequence which are suspected to be contributing for low yield of the protein and substituting the Native codons of the genome with optimized codons. This was done with one amino acid at one time. The expression levels and other properties were analyzed and were compared to the master optimized sequence. If improved expression levels are obtained, then such sequence was used for carrying out further modifications. If further modifications into the sequence results into reduction in the yields of protein or affects other properties of the protein then such modification was avoided. Many such cycles were followed to optimize the sequence.

(15) The flanking restriction sites, Ndel and Xhol were included at the termini of the gene. Following digestion of the synthetic DNA with the restriction enzymes Ndel and Xhol, the 0.503 Kb gene was then ligated via T4 DNA ligase into pBR 322 derived plasmid vector (FIG. 3, FIG. 4), which was also digested with these two enzymes. The recombinant plasmid was then introduced into E. coli strain BL21 (DE3) by transformation (1). The transformation mixture was plated on LB agar plates containing kanamycin (75 micrograms per ml) to allow for selection of colonies containing the pBR 322 derived plasmid/IF-alpha-2a (designated plasmid No. 1). Isolated colonies were further purified by plating and analyzed for IPTG inducible gene expression by standard methods.

Example 2: Comparison of Expression Yields Achieved Using Codon Optimized cDNA and Native cDNA

(16) The E. coli cells transformed with the vector containing codon optimized cDNA and vector containing native cDNA were grown on the media. The growing cells were subjected to IPTG inducible gene expression and the protein produced was analyzed using SDS PAGE analysis. The yield achieved for expression of the protein using codon optimized cDNA was 10 fold more than the yield achieved using native sequence of IFN alpha-2a as confirmed by SDS PAGE analysis and IMAGE Quant (GE healthcare) (FIGS. 5A and 5B).