Optimization of affinity-based tubulin purification and polymerization kinetics from goat brain and mung seedlings

Abstract

Microtubules (MT) form cytoskeletal polymers consisting of α− and β−tubulin dimers. Understanding MT polymerization kinetics can shed light on intrinsic physiological properties and diversification (Jain et al., 2021). In previous work, GST-TOG-1/2 column-based affinity purification was devised by Widlund et al. (2012), as an alternative to activity-based purification. Here, I report the optimization of expression and purification conditions for GST-TOG-1/2 protein, make a column and test the binding efficiency of an existing one and measure the effect of crowdants on goat brain and mung tubulin polymerization kinetics. The TOG-1/2 protein was successfully overexpressed in Escherichia coli BL21-DE3 Rosetta cells from a plasmid pGEX-6P-1. Complexation of 5.7 mg of purified TOG-1/2 with 1 ml of NHS-activated sepharose resin resulted in very poor binding, which when tested with BSA was found to be due to lack of activity of the NHS-sepharose material. As an alternative, I purified tubulin using a pre-existing column and found 23 to 48% elution of goat brain tubulin depending on column volume and salt used- either ammonium sulphate ((NH4)2SO4) or potassium chloride (KCl). Polymerization kinetics of goat brain and mung seedling tubulin was performed to optimize conditions for activity testing. Glycerol as a crowdant was tested on goat brain and mung tubulin, and the effect of 10% and 15% glycerol increased polymerization for goat tubulin and slightly for mung tubulin. Thus, I have optimized the isolation of GST-TOG-1/2, attempted to make an affinity chromatography column established the elution conditions of previously prepared affinity columns and optimized conditions for polymerization kinetics of goat brain and mung tubulin in the presence of crowdants.