Purification and Characterization of Protein and Protein Complexes
Instructors – Yongna Xing and Richard Burgess
Description: This is a 2-credit lecture course with a short-term, intense format that meets from 1:20-3:15 PM on Tues., and Thurs. for eight weeks before and after Spring Recess of 2022. The classroom will be room 125 old McArdle Building. This course will be given in the spring of every other year, 2022, 2024, etc. It is primarily a lecture course consisting of 26 lectures with two lectures per day, two half-semester exams, some take-home problems, and a term paper on a topic relating to the course. For the term paper, the students are asked to read at least twelve articles and prepare an informative summary of the topic chosen with references.
Important new features include 1) protein preparation and assay development for drug discovery, and protein-chemical interactions; 2) Cryo-EM for determining high-resolution structure of protein complexes, particularly oligomeric macromolecular complexes, which has increasing advantage over X-ray crystallography and NMR; 3) development of antibody, nanobody, and Fab for proteins of interest.
Goals of the course are: 1) to introduce the most important and useful concepts of protein purification and handling, 2) to help students to develop an intuition about how to work with proteins- so that they can “think like a protein”, 3) to introduce useful, modern tools for characterizing protein structure and function, and 4) to guide students to ongoing sources of information and resources. Students are also encouraged to discuss their research problems and seek solutions from the knowledge learned in the course and from discussion with other course participants and instructors.
Lecture topics include: Introduction-Protein purification overview; Properties of proteins/types of separation methods; Assays – following an enzyme through a purification; Protein characterization; Protein inactivation and stabilization/solution components; Purification strategy/starting materials/preparing cell/tissue extracts; Precipitation methods; Phase partitioning; Dialysis, desalting, concentration, and ultrafiltration; Preparative electrophoresis, chromatofocusing, isoelectric focusing, capillary electrophoresis; Purification of membrane proteins/glycoproteins; Column chromatography – theory and concepts; Sizing – gel filtration chromatography; Ion exchange, Affinity, Immunoaffinity, and DNA affinity chromatography; HPLC: Columns and hardware, theory, methods development, applications; Micropurification by eluting from SDS gels; Overproduction of cloned gene products; Purification and refolding of insoluble overproduced proteins; Engineering proteins for ease of purification and characterization; Recent advances in studying protein-protein interactions; isolation, assembly, and characterization of protein complexes; Proteomics/protein microarrays; Protein characterization methods; Post-translational modifications and characterization with mass spectrometry; Protein structure determination by X-ray crystallography, NMR, and Cryo-EM; Protein engineering and generation of antibody, nanobody and Fab for elucidating protein structure and function; Drug discovery, small molecules and chemical mimics in protein structure and function.
This course is usually taken for credit by about 10-20 graduate students, and occasionally by senior undergraduates. Auditors or those who wish to sit in on part or all of the lectures are welcome (usually as many as a dozen specialists, advanced graduate students, postdocs, and faculty sit in).
For more info contact: Yongna Xing – email@example.com (262-8376 (o); 848-219-5646 (cell)) or Dick Burgess – firstname.lastname@example.org (263-2635 (o); 608-271-9335 (h))
Undergraduate and graduate students in this course will be graded on a separate curve, which is in compliance with the Higher Learning Commission’s requirements for graduate courses with undergraduate participants.