Question

Let’s assume there is a myosin transport protein complex that you would like to purify. The complex normally exists in yeast (in very small amounts) where myosin is bound to 2 other proteins via multiple weak interactions that include ionic interactions and hydrogen bonds. Using a recombinant plasmid with the myosin gene of interest inserted in a yeast vector (that can express the gene of interest in yeast cells), design an experiment to purify the transport complex of interest that includes the two proteins bound to myosin.

Answer 1

Ans)

step1: To purify the transport complex ( two proteins bound to mysoin) were overexpressed by inducing certain strain in medium for 48 hours

step2: Purification done by S-tagged

step3: lyophilized cells were resuspended in extraction buffer

step4: cell extract were incubated with covalently coated with anti FLAG antibody at 4 degree for 30 min

step5: to collect supernatant concentrated to gel filtration by using protein concentrator

step 6: estimating the size of complex and separated by using analytical ultracentrifugation