Cambridge Healthtech Institute recently interviewed Dr. William Clay Brown of University of Michigan about Flavivirus NS1. He will present on “Using High-Throughput Techniques to Produce Difficult Targets: Flavivirus NS1, an Updated Case Study” at the Sixth Annual Higher-Throughput Protein Production and Characterization, taking place January 12-13, 2017 as part of the 16th Annual PepTalk event which runs from January 9-13, 2017 in San Diego, CA.

How did you develop a robust production pipeline for NS1 protein, a multi-functional virulence factor, from several different Flavivirus?

By evaluating many constructs, expression conditions and buffer compositions using high-throughput methodology we were able to identify the best combinations for producing NS1 protein from Dengue 2, West Nile and Zika virus. Once the production and purification conditions were identified applying them to obtain sufficient protein for crystallography and biochemical characterization was very straight-forward.

What is the importance of determining crystal structures of full-length NS1?

The structure of the NS1 protein reveals two distinct “faces” to the protein, one extended hydrophobic surface and one more charged surface. Having the structure of the NS1 proteins has allowed us to infer how the protein interacts with membranes through its hydrophobic face while still inside the cell. It has suggested the mechanism for how NS1 interacts and is essential for viral replication even though NS1 is on the opposite side of the endoplasmic reticulum from the rest of the flavivirus replication machinery. It has been used to guide mutagenesis experiments that are further defining which parts of the protein are involved in its various functions. The hexameric structure shows the hydrophobic face to be internal and the more charged surface to be external where it can interact with complement proteins in the circulatory system. Comparisons of surface charge differences between the NS1 proteins from different flavivirus may be useful in the development of diagnostic tools for determining which virus is involved in a particular infection, something that has been difficult in the past. It may also lead to the development of inhibitors or vaccines that may be used to treat flavivirus infection.

What affect does glycosylation have on NS1 and its functioning?

Mutations that remove the glycosylation sites or production in glycosylation deficient hosts have shown that glycosylation is involved in protein stability, secretion as a hexamer and interactions of secreted NS1 with proteins of the complement system. Some studies have also suggested reduced glycosylation of NS1 is responsible for reduced RNA replication and production of less infective viral particles probably through reduced stability and loss of NS1 interaction with the replication machinery.

What are you most looking forward to at PepTalk 2017?

I am looking forward to hearing talks on the development of cutting-edge techniques in protein production and purification, and interacting with top-level scientists from industry and academics who share my interests.


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