Open AccessAutomation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies
Author(s) -
Bairy Sneha,
Gopalan Lakshmi Narayanan,
Setty Thanuja Gangi,
Srinivasachari Sathya,
Manjunath Lavanyaa,
Kumar Jay Prakash,
Guntupalli Sai R,
Bose Sucharita,
Nayak Vinod,
Ghosh Swagatha,
Sathyanarayanan Nitish,
CaingCarlsson Rhawnie,
Wahlgren Weixiao Yuan,
Friemann Rosmarie,
Ramaswamy S.,
Neerathilingam Muniasamy
Publication year - 2018
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.13041
Subject(s) - cloning (programming) , sialic acid , computational biology , expression vector , biology , escherichia coli , biochemistry , recombinant dna , gene , computer science , programming language
Summary 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.