2 - Recombinant DNA and Protein Technology

Summary

Introduction

To understand cellular structures and their functions, comprehensive knowledge of proteins and the mechanisms of their action is needed. For better understanding of cellular processes, the protein component needs to be extracted from a complex mixture. Earlier, proteins were extracted in a purified form either by using large amounts of animal or (a variety of) plant tissues or biological fluids. With the advent of recombinant protein technology, most biochemical projects start with conceptualizing purification of the target protein in a recombinant form. The capabilities of this technology allows biochemists to design, clone, extract, and purify a protein for its biochemical characterization, better understanding, and research/commercial applications.

Protein purification is a series of steps required to isolate a target protein from a complex mixture of biomolecules. Protein purification may be preparative or analytical. Preparative purification aims to produce relatively large amount of protein for their use, such as soy protein extract and insulin. Analytical purification aims at obtaining just sufficient amounts of protein required for the specific research. In general, recombinant protein purification steps are straightforward. The process starts from identifying the gene of interest, which is a clone and transform into a proper host, and after induction of gene expression the designed protein is ready for further experimentation, such as development of purification protocols and its biochemical characterizations. However, these experiments involve several steps that can go wrong, such as inappropriate transformation, poor growth conditions, inclusion body formation, degradation of the target protein, inactive protein, and even no expression.

Inside tissue, some proteins are present in enough amounts that can be isolated from their host directly. But for getting larger amounts of proteins, scientists need to enhance their expression by using an appropriate host and develop protocols for their purification. There are four main steps involved: a gene of interest, an optimized vector that contains that gene, an appropriate expression host, and a complete purification procedure. Cloning, expression, and purification are the three major steps in getting the desired amount of pure protein.

Cloning

Gene cloning employs a series of molecular biology methods that culminate with the insertion of the gene of interest into a host cell and replicating this in many other cells under appropriate conditions of the expression of that gene.