TY - JOUR T1 - Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. JF - Microb Biotechnol Y1 - 2018 A1 - Bairy, Sneha A1 - Gopalan, Lakshmi Narayanan A1 - Setty, Thanuja Gangi A1 - Srinivasachari, Sathya A1 - Manjunath, Lavanyaa A1 - Kumar, Jay Prakash A1 - Guntupalli, Sai R A1 - Bose, Sucharita A1 - Nayak, Vinod A1 - Ghosh, Swagatha A1 - Sathyanarayanan, Nitish A1 - Caing-Carlsson, Rhawnie A1 - Wahlgren, Weixiao Yuan A1 - Friemann, Rosmarie A1 - Ramaswamy, S A1 - Neerathilingam, Muniasamy AB -

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

VL - 11 IS - 2 ER -