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

Microb Biotechnol. 2018 Mar;11(2):420-428. doi: 10.1111/1751-7915.13041. Epub 2018 Jan 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation, Laboratory / methods*
  • Cloning, Molecular / methods*
  • Enzymes / genetics
  • Enzymes / isolation & purification*
  • Enzymes / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression*
  • Genetic Testing / methods
  • Metabolic Networks and Pathways / genetics*
  • N-Acetylneuraminic Acid / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Enzymes
  • Recombinant Proteins
  • N-Acetylneuraminic Acid