Webinar Highlights

You will learn:

  • Introduction to the structure and function of the tuberous sclerosis protein complex (pTSC)
  • A novel strategy for isolating and purifying protein complexes from native sources using recombinantly produced Fabs
  • How Next Generation Protein Sequencing (NGPS) works
  • Applications of NGPS to aid design and engineering of recombinant antibody fragments

Abstract

The tuberous sclerosis protein complex (pTSC) is an intracellular stress-sensor that nucleates a proteinaceous signaling hub to integrate regulatory information about the growth and energy consumption of the cell. The structure and stoichiometry of pTSC have been revealed through previous biochemical and cryo-electron microscopy (cryo-EM) studies, hinting at the possibility of pTSC forming large higher-order oligomers. However, these studies required the stabilization of the protein with chemical cross-linking or graphene oxide-coated grids during sample preparation, making it difficult to obtain a complete understanding of its architecture.

Though there has been immense progress in filling information gaps, there is still much to be learned with regards to understudied and/or unstable proteins and protein complexes, like pTSC. One of the major challenges that researchers often face is the effort to express, purify, and isolate proteins for downstream structural characterization. Purification and expression of recombinant proteins remain the preferred method for obtaining protein samples; however, this can be a demanding task for certain proteins.

Protein complexes may form higher-order structures only when they are expressed under native conditions within the host organism. Therefore, novel techniques for isolating proteins from their native sources allow for progress in characterizing those unstable, ‘tricky’ proteins. Next generation protein sequencing (NGPS) is one approach that is being leveraged in protein purification strategies. With NGPS, the primary amino acid sequences of commercially available mAbs are now being derived and utilized to engineer recombinant antibodies and antibody fragments that show improved target specificity for capturing proteins and protein complexes from their native sources. Strategies like these can help provide the structural and functional information towards our understanding of diverse proteins and their intricacies.

Speaker Bios

David L. Dai, PhD Candidate

University of Toronto

PhD Candidate

David obtained his BSc from McGill University majoring in immunology (hons) in 2019 before joining Dr. Mohammad Mazhab-Jafari’s lab in the Department of Medical Biophysics at the University of Toronto as a direct-entry PhD student. He is now a 4th year PhD candidate whose research focuses on purifying and determining the atomic structures of human signaling proteins relevant to cancer using cryo-electron microscopy (cryoEM).

For his efforts in his doctoral studies so far, David has received Ontario Graduate Scholarships in 2020 and 2021, and the NSERC Canada Graduate Scholarships Doctoral Program (CGS-D) in 2022. His research has been published in multiple peer-reviewed journals with the focus of his talk today having been featured in ACS Biochemistry.

 

Monique Seymour, MSc

Rapid Novor

Sr. Scientific Sales Executive

Monique Seymour graduated from the University of Glasgow with a Master’s in Biotechnology and Management. She has years of experience working in the proteomics industry, working with scientists in both Diagnostic and Therapeutic areas to mitigate risk and speed up discovery using next generation protein sequencing and other innovative technologies. Monique is passionate about furthering life sciences research to improve global patient health.

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