How to make recombinant protein has revolutionized the field of biochemistry. When a researcher starts a new project that will require purified protein, he or she immediately starts thinking about how to make it. The days when kilos of animal and plant tissues or large volumes of biological fluids were required for the purification of small quantities of a given protein are almost gone.

However, to make recombinant protein a success requires a great deal of time and expertise. Often, it takes years to develop protocols that produce proteins with the chemical and physical homogeneity necessary for clinical use or structural determinations.

How to Make Recombinant Protein: An In-Depth Guide

The first step to making a recombinant protein is to select the best system to express the desired protein. This is determined by the size of the gene encoding for the protein, functional activity requirements, and optimum yield of the recombinant protein.

Once the desired system is selected, the protein-coding gene must be inserted into the host cell. The next step is to induce protein expression by adding a peptide activator or a small molecule that blocks transcription and translation. This is typically done in a high-density cell paste that contains a variety of other cells such as Escherichia coli, Saccharomyces cerevisiae, or mammalian cells like baby hamster kidney (CHO) or Chinese hamster ovary (CHOC).

The final step is to perform a one-dimensional SDS-PAGE to determine the amount of recombinant protein in the cell paste. If a Coomassie blue-stained band corresponding to the expressed protein is observed, the expression has been successful.