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Saturday, March 17, 2012

Shake Flask Expression Studies / Shake Flask Protein Expression and Analysis

Shake flask expression studies are the initial phase for screening of recombinant clones. Shake flask expression studies helps to pick the clone bearing colony with highest yield. Colony PCR can be used to check the whether the colony has been transformed with the gene of interest. Colonies which has grown on selective media after transformation can be taken for shake flask expression studies. positive clones (colonies bearing gene of interest) can be selected by doing colony PCR. generally shake flask expression is done initially on smaller volumes like 5-10 ml in small screw cap tubes or in conical flasks. A single colony is picked and innoculated into the suitable media with selectable markers (generally antibiotics) depending on the plasmid it harbours. Once the OD reaches 1 induction can be started by adding suitable inducer. Incase of E.coli strains IPTG is used as inducer for the expression. 0.5mM to 1mM can be used to induce the gene. Innoculated flasks or tubes are kept on shaker incubator, higher RPM is recommended for good induction.depending on the organism used either bacteria or yeast suitable temparature need to be maintained.

Shake Flak Culture

IPTG acts an inducer, the principle involved in Induction using IPTG is, Expression of many proteins in bacteria is controlled by the Lac Operon. An operon is a collection of linked genes under common, coordinate control. Typically bacteria do not use lactose as a source for food, however when enough lactose is added to the cells, lactose binds to repressor proteins and will cause the induction of the production of two different proteins, permease, used to transport carbohydrate and ß-galactosidase. ß-galactosidase will hydrolyze the disaccharide lactose to the monosaccharides glucose and galactose, then the bacteria can continue to grow. Many researchers and companies have removed these two genes and placed a gene for a protein that they want. This way, when lactose or its non-metabolized inducer isopropyl beta-thiogalactoside, (IPTG), is added the cell is sort of tricked into making the protein cloned into the lac operon rather than permease and ß-galactosidase.

Induction of the cloned gene is different in bacteria and yeast so suitable inducer and its concentration need to be maintained for getting higher expression levels.

Induced samples were taken at regular intervals (0hr, 6hr, 12hr, 24hr, etc) and can be analyzed on SDS-PAGE gel and HPLC, depending on the expressed protein gel percentage can be made. An uninduced sample and a protein marker of suitable range is also loaded. .Both HPLC and gel gives the expression rates.The clone which produces high amount of protein is selected and used for scale up. Biochemical Protein Assays can be done to quantify the expressed protein. Protein characterization need to be done for the expressed protein which will help in Downstream processing and further protein purification Steps.