Key information

Next planned course: Fall 2021
Location: UiB
Course responsible: Øyvind Halskau

Grading: Evaluation of lab reports (pass/fail)
Credits: 3 ECTS  


Registration

Students should register to BioCat, as well as:

Content

The course will teach how to plan and execute a protein/enzyme expression and purification project. The course will involve the use of relevant bioinformatics tools, primer design and gene optimization. There will be opportunities for discussing and supporting the student’s own project. Focus will be on rational project design, to ensure effective use of time. The course will teach how to optimize new expression/purification systems with respect to different conditions, bacterial strains and use of fusion proteins for purification and solubilisation of the target protein. Functional fusion partners with enzymatic activities, like DsbC, will also be discussed. Troubleshooting of common problems will be discussed both practically and theoretically. The course will involve mutagenesis of an enzyme active site, as well as the use of minimal and other medias for isotopic labelling. Use of NMR will be presented very superficially as a way of assessing protein fold (i.e. HSQC fingerprinting). Activity measurement of selected enzyme products will be taught and performed, also using NMR.

Teaching

  • 1 day of individual student preparation using material provided, to be completed before the course starts
  • 5 days of laboratory and lecture work before and after lunch
TimeMondayTuesdayWednesdayThursdayFriday
9.00-12.30Lab: Primer design Lab: Harvest cells Lectures and Lab workLectures and Lab workLectures and Lab work
13.00-17.00Lab: Create competent cells. Overnight growthLab: Affinity purificationLectures and Lab workLectures and Lab workLectures and Lab work
Schedule
  • Preparation using material made available prior to course 
  • Working on planning own project – prior to and after course 
  • Laborarory report

Learning outcome

The course aims to give an introduction and basic skills in recombinant protein/enzyme production, purification and characterization.

The course participants will be guided in the planning of expression and purification projects, primer design for PCR cloning, and protein expression in E.coli. The course will focus upon optimization of the various steps in the experimental procedures and focus upon trouble shooting.

The candidate will, after completing the course, have:

Knowledge within

  • Primer design and gene optimization
  • Insights in various trouble shooting problems and strategies to solve them
  • Insight in medium throughput cloning and protein expression in E. coli
  • Simple application of NMR to assess protein fold after purification
  • Co-expression and purification strategies
  • Insight in mutagenesis techniques

Skills in

  • Cloning of genes into vectors containing different tags and fusion proteins
  • Use of bioinformatics in analysis and planning of a protein expression project
  • Protein expression and mini screen of the expression
  • Assess different E.coli strains, growth conditions and fusion partners to optimize the quality of expressed protein
  • Simple enzymatic activity assays

General competence in

  • Rational project planning
  • Use of fusion protein- and alternative purification strategies
  • Scale-up of positive candidates to a protein midi-preparation using purification machines