Methods in Biochemistry

Using bacterial cells to produce proteins

The first step was to prepare an active protease. For this purpose, I used bacterial cells Escherichia coli NiCo21 which can take up a foreign plasmid (circular DNA) and start producing the protein encoded; this process is called transformation. The cells are allowed to grow in a nutritious medium and, subsequently, harvested by centrifugation.

Isolating proteins with a nickel column

The plasmid with the protein of interest contained much other information, including a sequence coding for six histidine molecules. These were added to enable isolation of the protein via a nickel column which has high affinity towards the histidine-tag. But first, the bacterial cells must have been lysed. To achieve this, I use sonication, which applies high frequencies of sound to disrupt bacteria. Afterwards, I employ centrifugation to remove the insoluble cellular components. Then I subject the soluble part called supernatant to the nickel column.

Purifying proteins

There is an adage: “Never waste pure thoughts on an impure protein.” Indeed, I conduct at least two methods to purify my proteins. Usually, those are the size-exclusion chromatography, based on separating proteins based on size, and the ion-exchange chromatography, separating proteins based on charge. I conduct both in a sophisticated FPLC (fast protein liquid chromatography) system.