title: A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform.
abstract: The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.
authors.affiliation: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE. g.ordidge@ucl.ac.uk
authors.forename: Gemma C
authors.initials: GC
authors.lastname: Ordidge
authors.forename: Gareth
authors.initials: G
authors.lastname: Mannall
authors.forename: John
authors.initials: J
authors.lastname: Liddell
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
authors.forename: Martina
authors.initials: M
authors.lastname: Micheletti
mesh_terms: D000818:Animals; D002645:Chickens; D057166:High-Throughput Screening Assays / Q000379:methods*; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry*; D058849:Protein Refolding; D012371:Robotics / Q000379:methods*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D009113:Muramidase