Protein Expression & Scale up

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GENERAL INTRODUCTION

Proteins are instrumental in all biochemical transformations that occur in any living system. They perform diverse functions such as transport, transformation, stimulation, regulation, stabilization and host of other functions. The information for the protein structure is stored in genes (DNA). Any change in DNA structure can result in the structural changes in proteins. There genetic diseases due to mutations in genes are known. There are diseases due to by destruction of certain cells. To help ameliorate genetic defect and infectional destructions of cells that produce specific proteins, isolation of a specific gene and its expression is important. To make it commercially available like the productions of insulin, scale-up of the process is important.

DESCRIPTION

Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains the template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. The selection of the system depends on the type of protein.

Bacterial protein expression systems are popular because bacteria are easy to culture, grow fast and produce high yields of recombinant protein. However, multi-domain eukaryotic proteins expressed in bacteria often are non-functional because the cells are not equipped to accomplish the required post-translational modifications or molecular folding. Also, many proteins become insoluble as inclusion bodies that are very difficult to recover.

Mammalian in vivo expression systems usually produce functional protein, but the yield is low, cost of production is high and mammalian cell culturing is time-consuming. In addition, in vivo systems are not conducive to either high throughput protein synthesis or expression of proteins that are toxic to host cells.

Cell-free protein expression is the in vitro synthesis of protein using translation-compatible extracts of whole cells. In principle, whole cell extracts contain all the macromolecular components needed for transcription, translation and even post-translational modification. These components include RNA polymerase, regulatory protein factors, transcription factors, ribosomes, and tRNA. When supplemented with cofactors, nucleotides and the specific gene template, these extracts can synthesize proteins of interest in a few hours.

APPLICATION

• Production of hormones – Insulin
• Production of proteins for constituting physiological fluids such as blood
• Production of commercially important enzymes
• Mutation analysis
• Genetic variation and detection
• Enzymatic activity analysis
• Molecular diagnostics
• Post-translational modification analysis

MAJOR INDUSTRIES AND TOP PLAYERS IN INDIA

• Serum Institute of India
• Biocon Pvt Ltd
• Panacea Biotech
• Shantha Biotechnics
• Novozymes South Asia
• Advanced Enzyme Technologies
• Dr. Reddy’s Lab

FUTURE PROSPECTS

The production of recombinant proteins has great potential as effective tool for research and commercial biotechnology. During the last few years, there has been an increasing interest in the field of gene regulation and protein synthesis in different systems that may be used as a novel approach for production of some important therapeutic drugs or proteins.

Recent improvements/developments in this area have significantly increased its utility and enabled various groups to explore the possibilities of production of recombinant proteins, which can be directly or indirectly used for various pharmaceuticals, nutraceuticals and therapeutic purposes.

Commercial production of proteins is required in the development of drugs, pharmaceuticals, nutraceuticals and enzymes of commercial importance.