%0 Journal Article %J Microb Biotechnol %D 2018 %T Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. %A Bairy, Sneha %A Gopalan, Lakshmi Narayanan %A Setty, Thanuja Gangi %A Srinivasachari, Sathya %A Manjunath, Lavanyaa %A Kumar, Jay Prakash %A Guntupalli, Sai R %A Bose, Sucharita %A Nayak, Vinod %A Ghosh, Swagatha %A Sathyanarayanan, Nitish %A Caing-Carlsson, Rhawnie %A Wahlgren, Weixiao Yuan %A Friemann, Rosmarie %A Ramaswamy, S %A Neerathilingam, Muniasamy %X

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.

%B Microb Biotechnol %V 11 %P 420-428 %8 2018 Mar %G eng %N 2 %R 10.1111/1751-7915.13041 %0 Journal Article %J Acta Crystallogr F Struct Biol Commun %D 2017 %T Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. %A Caing-Carlsson, Rhawnie %A Goyal, Parveen %A Sharma, Amit %A Ghosh, Swagatha %A Setty, Thanuja Gangi %A North, Rachel A %A Friemann, Rosmarie %A Ramaswamy, S %K Adenosine Triphosphate %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Cloning, Molecular %K Crystallography, X-Ray %K Escherichia coli %K Fusobacterium nucleatum %K Gene Expression %K Genetic Vectors %K Hexosamines %K Models, Molecular %K Phosphotransferases (Alcohol Group Acceptor) %K Protein Binding %K Protein Conformation, alpha-Helical %K Protein Conformation, beta-Strand %K Protein Interaction Domains and Motifs %K Protein Multimerization %K Recombinant Proteins %K Sequence Alignment %K Sequence Homology, Amino Acid %K Substrate Specificity %X

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

%B Acta Crystallogr F Struct Biol Commun %V 73 %P 356-362 %8 2017 Jun 01 %G eng %N Pt 6 %R 10.1107/S2053230X17007439