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

Sneha Bairy,Rosmarie Friemann,S Ramaswamy,Sathya Srinivasachari,Muniasamy Neerathilingam,Nitish Sathyanarayanan,Jay Prakash Kumar,Lavanyaa Manjunath,Lakshmi Narayanan Gopalan,Thanuja Gangi Setty,Vinod Nayak,Weixiao Yuan Wahlgren,Swagatha Ghosh,Sai R Guntupalli,Sucharita Bose,Rhawnie Caing‐Carlsson

doi:10.1111/1751-7915.13041

Abstract

SummaryThe 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.

Highlights

We use a high-throughput platform to perform cloning, expression and purification of eighteen enzymes involved in sialic acid catabolism and surface sialylation by four different Gram-negative bacteria (Haemophilus influenza, Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholera) (Table S1)
Our main objective was to clone the genes involved in sialic acid catabolism and the surface sialylation pathway into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo to get the gene products in soluble form
Primers were designed for each of the genes to amplify the genes with 15 bp of the attB sites for cloning into the Gateway vector – pET300/NT-DEST – containing an N-terminal 69 His tag

Summary

Introduction

The revolution and the reduction in the cost of DNA sequencing technologies have led to large numbers of protein sequences being determined; their structures are being determined at a much lower rate. Another automation platform for cloning, expression and purification is described (Mlynek et al, 2014), where some steps such as PCR, plasmid extraction and expression are automated, but would still require manual interventions at multiple points such as gel extraction, transformation and culture inoculation. A cell-free expression-based method for high-throughput cloning and expression has been developed (Betton, 2004); steps such as protein and DNA analysis are not automated.

Results

Conclusion