Expression of a partial synthetic human TNF cDNA in E. coli

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Engineering

Background:

• Tumor Necrosis Factor (TNF) is a crucial cytokine involved in inflammatory responses.
• Production of recombinant TNF (rhTNF) is essential for therapeutic and research applications.
• Efficient expression systems are needed for large-scale rhTNF production.

Purpose of the Study:

• To construct and express a recombinant human tumor necrosis factor (rhTNF) cDNA.
• To develop a scalable method for producing biologically active rhTNF.
• To characterize the purity and identity of the produced rhTNF.

Main Methods:

• Isolation of the human TNF gene from a genomic library.
• Construction of a partial synthetic rhTNF cDNA by ligating a major portion of the fourth exon with a synthesized DNA fragment.
• Subcloning into a vector for expression in E. coli, utilizing a 5-1 fermentator for large-scale production.
• Purification using High-Performance Liquid Chromatography (HPLC) and characterization of the N-terminal amino acid sequence.

Main Results:

• Successful expression of rhTNF in E. coli, yielding significant bacterial pellet and high cytotoxicity units per milliliter.
• Purification of rhTNF to approximately 95% purity, obtained as a white powder.
• Determination of the N-terminal amino acid sequence, confirming identity with natural human TNF.

Conclusions:

• A viable method for producing high-purity, biologically active rhTNF using a synthetic cDNA approach in E. coli has been established.
• The recombinant protein is functionally and structurally equivalent to the natural human TNF.
• This production strategy holds promise for the large-scale manufacturing of rhTNF for potential therapeutic uses.