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Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.

Artikel i vetenskaplig tidskrift
Författare Sneha Bairy
Lakshmi Narayanan Gopalan
Thanuja Gangi Setty
Sathya Srinivasachari
Lavanyaa Manjunath
Jay Prakash Kumar
Sai R Guntupalli
Sucharita Bose
Vinod Nayak
Swagatha Ghosh
Nitish Sathyanarayanan
Rhawnie Caing Carlsson
Weixiao Yuan Wahlgren
Rosmarie Friemann
S Ramaswamy
Muniasamy Neerathilingam
Publicerad i Microbial biotechnology
Volym 11
Nummer/häfte 2
Sidor 420-428
ISSN 1751-7915
Publiceringsår 2018
Publicerad vid Institutionen för kemi och molekylärbiologi
CARe - Centrum för antibiotikaresistensforskning
Sidor 420-428
Språk en
Länkar dx.doi.org/10.1111/1751-7915.13041
www.ncbi.nlm.nih.gov/entrez/query.f...
https://gup.ub.gu.se/file/207323
Ämneskategorier Biokemi och molekylärbiologi

Sammanfattning

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.

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