AsianScientist (Nov. 20, 2017) – A team from the NUS Yong Loo Lin School of Medicine (NUS Medicine) has invented a fundamentally new way of folding and protecting recombinant proteins. This protein-in-a-protein technology, which can improve functional protein yields by 100-fold and protect recombinant proteins from heat, harsh chemicals and proteolysis, has been described in a paper published in Nature Communications.
The expression and stabilization of recombinant proteins is the cornerstone of the biologics and pharmaceutical industries. The costs and complexity associated with manufacturing difficult-to-fold recombinant proteins at an industrial scale are a significant limiting factor to their use in clinical and industrial applications.
In the present study, Assistant Professor Chester Drum of NUS Medicine engineered a 12-nanometre diameter exoshell and wrapped it around a protein of interest (POI). They showed for the first time that this technology can be used to fold and protect a variety of proteins within engineered cavities that are less than 1:10,000 the width of a human hair.
The researchers developed this protein-within-a-protein technology with the help of Archeoglobus fulgidus, a hardy bacteria that is naturally found in hydrothermal vents. These hyper-thermophilic bacteria have evolved unique solutions for protein folding and stabilization due to the extreme environments in which they live.
In particular, the researchers made use of an iron-carrying, 24-subunit protein in A. fulgidus called ferritin, whose natural function is to store and carry iron in the blood. Ferritin from A. fulgidus has two unique properties: first, four tiny pores in its shell provide small molecules access into the cavity; second, unlike human ferritin which is stable at low salt concentrations, the engineered A. fulgidus ferritin dissociates at low salt concentrations, allowing the contents of the cavity to be released by a simple pH switch from 8.0 to 5.8. Once dissociated, the POI can be released enzymatically.
To demonstrate the wide versatility of their technology, the researchers tested their exoshell technology by fusing one of the 24 ferritin subunits around three POIs with diverse properties—green fluorescent protein, horseradish peroxidase (HRP) and Renilla luciferase.
Not only did the exoshell help increase the yields of all three POIs, the researchers were also able to deliver cofactors heme and calcium, in addition to oxidizing conditions, to ensure that complex POIs such as HRP protein could fold and function properly.
Besides helping to fold the POIs correctly, the exoshells were also protective against a wide range of denaturants, including high concentration trypsin; organic solvents such as acetonitrile and methanol; and denaturants such as urea, guanidine hydrochloric acid, and heat.
“We hypothesize that the significant increase in functional protein yield may be due to the complementation between the negatively charged proteins and the positively charged exoshell internal surface,” said Drum, who is also a consultant cardiologist at the National University Hospital and director of the Clinical Trial Innovation Lab at TLGM, A*STAR.
“Our findings highlight the potential of using highly engineered nanometer-sized shells as a synthetic biology tool to dramatically affect the production and stability of recombinant proteins.”
Drum’s current research bridges the gap between basic biochemistry and clinical care. He is currently the primary investigator on a multi-institutional, 3,000-person observational trial in Singapore that studies how personalized drug metabolism affects drug response.
The article can be found at: Deshpande et al. (2017) Thermostable Exoshells Fold and Stabilize Recombinant Proteins.
Disclosure: Dr. Juliana Chan, the editor-in-chief of Asian Scientist Magazine, is a co-author on this paper. Asst Prof Chester Drum, the corresponding author on this paper, is a non-executive director of Wildtype Media Group Pte Ltd, the company that publishes Asian Scientist Magazine.
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Source: National University of Singapore; Photo: Chester L. Drum.
Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.
