Application of rDNA Technology and Genetic Engineering in the Production of Interferon

Interferons are low molecular weight proteins produced by virus infected cells, which in non-infected cells can induce the formation of a second protein. This protein has the ability to prevent transcription of any viral mRNA produced in that cell. Interferons exhibit antiviral activity, anti-proliferative activity, and many effects on the immune system.

Interferons on the basis of their source, biological, physicochemical and antigenic properties are grouped into the following three classes:

  1. IFN- α (leukocytes),
  2. IFN- β (fibroblasts), and
  3. IFN- r (immune).

IFN- α can be effectively used in the treatment of some melanomas and renal cell cancers, while IFN- β can be used in solid tumours. Trials of IFN- γ have proved it a potential stimulator of the immune system with enhanced anti-tumour properties. IFN- α with 865 nucleotide (166 amino acids) sequences and IFN- β with 836 nucleotide sequences can be expressed in E. coli. The genomic DNA – sequences of both IFN- α and IFN- β lack introns; while IFN- γ codes for 146 amino acids and include introns. Production of interferons encoded by DNA sources involves the synthesis of cDNA to the mRNA isolated from a cell line, tissue or organ followed by screening and selection of cDNA clone.

Synthesis of interferon gene can also be done chemically if the sequence of the gene encoding that particular interferon is known. Thus, a variety of interferon molecules, IFN- α (IFN- α1, IFN- α2, IFN- αA, IFN- αB, IFN- αC), IFN- β and IFN- γ, can be produced. Synthetic genes have the advantage over the natural gene sequence for high level expression in microorganisms.

IFN synthesis in bacteria depends on the transcription of genes to mRNA and translation of mRNA to produce the protein. Transcription requires suitable promoters, adjacent operator region, ribosome binding site, and a start or initiation codon. In additional processes, the primary translated IFN is manipulated to functionally active IFN, the signal sequence is removed and glycosylated in case of IFN- β and IFN- γ. Intramolecular disulphide bridges are essential for the biological activity of IFN- α and IFN- β.

Human IFN – α

Synthesis of IFN- α1 gene involves assembly of 67 oligonucleotide fragments to give a double stranded molecule, in which each strand contains 514 base pairs. Natural gene sequence obtained by cDNA prepared from 125 fractions of mRNA from IFN producing human leukocytes can be cloned in E. coli using pBR322 vector. The cDNA inserted in the β-lactamase gene at the PstI site expresses a fusion protein. An mRNA hybridisation translation assay is used for identifying the cDNA clone containing an IFN- α.

The colonies are screened using 32p-labelled 320bp PstI fragments as probe. Constructed plasmids can be used for synthesising IFN molecules fused to β lactamase. Biologically active material hybrid with the IFN gene in three different reading frames can be produced. The various molecules of human IFN- α are expressed in E. coli, utilising different promoter or construct expression systems (β-lactamase, trip, lac UV5, lac, etc.).

Human myeloblastoid line KG-1 also serve as a source of mRNA for IFN coding sequences. A gene coding for a complete IFN- αA can be isolated after the formation of double stranded cDNA and cloning in E. coli. Inserting this gene in a plasmid vector (containing a part of the trp operon) results in a biologically active fusion product of amino acid and IFN- αA.

Human IFN- β

In an approach to form mRNA from cDNA, production of human IFN- β involves insertion of cDNA into pBR 322, followed by transformation into E. coli. Identification follows hybridisation to cDNA, enriched for IFN-containing sequences. The cDNAs hybridising to total mRNA (containing sequence for human IFN- β ) are isolated. Various molecules of human IFN- β in E. coli cloned from cDNA are expressed by incorporating various promoters or expression systems (lac or trp).

Human IFN- γ

The DNA fragment encoding IFN- γ with a sequence coded for a polypeptide of 166 amino acids (of which 20 constitutes signal sequence) can be produced from mRNA to cDNA approach. Bacterial clones can be screened from cDNA library by using 32-labelled cDNA probe prepared from either induced or unstimulated peripheral blood lymphocytes. cDNA can be expressed by transforming the fragment containing vector to E. coli.

A restriction site at codon-4 of the mature coding sequence is used for cleaving the single peptide. Two synthetic oligonucleotides are induced for restoring the codons or amino acids 1-4, incorporating an initiation codon ATG, with a restriction site cohesive terminus for joining a trp promoter. The product contains only a single gene, cross hybridising with IFN- γ-cDNA sequence.

Since the genomic DNA contains three introns, it cannot be expressed directly in prokaryotes; rather can be expressed from cDNA sequence in yeast cells and higher eukaryotic cells [like Ap8 monkey cells, Chinese Hamster Ovary (CHO cell line)]. Chemically synthesised gene for IFN- γ can be expressed in E. coli from a lac UV5 promoter. Anti IFN- γ serum can be used for neutralising antiviral cell lysate. IFNs can be purified by High Performance Liquid Chromatography (HPLC) or reverse phase liquid chromatography.

Read More Topics
Techniques used in recombinant DNA technology
Expression of cloned gene within the host
Vectors for preparing single stranded DNA

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Santhakumar Raja

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