Practical Course on rapid Cloning, Protein Expression and Purification, CPEP-2020 (3 ECTS), 29 January - 7 February 2020

Course information

Date: January 29th to February 7th, 2020.

Start: 09.30 January 29, 2020.

Place: Biochemistry teaching lab, KB.F4.04, KBC-building F, 4th floor (2 stairs up), Linnaeus väg 6

Time: about 8 h per day, mainly laboratory work but also lectures, tutorials, discussions & seminars.

Leader: Uwe Sauer, KBC, 070-635 1803, E-mail: uwe.sauer@umu.se    

Instructor: Gunter Stier, Heidelberg Univ., Germany, E-mail: gunter.stier@bzh.uni-heidelberg.de  

Registration deadline: Friday January 17, 2020. Admission on a first-come first-served basis.

Registeration form: see below

Participants: max. 16 (PhD students, post-docs or technical staff)

Credits: 3 ECTS on a pass – no pass basis. Full time participation is required to obtain a pass certificate.

Aims

After the CPEP course you should know how to:

• Plan and successfully execute your project. Bring your own project(s)*!!!
• Make efficient use of bioinformatics tools for planning and analysis of protein expression projects
• Design PCR primers and codon optimize genes
• Apply various fast cloning and mutagenesis techniques
• Use medium-throughput approaches for cloning and protein expression in E. coli
• Carry out fast parallel cloning of your gene(s) into vectors containing different tags and fusion proteins
• Use of fusion proteins and associated purification strategies
• Test for optimal protein production: different strains, growth conditions and fusion partners
• Co-express components of protein complexes and their purification strategies
• Prepare and use of auto-inducing medium
• Do protein expression mini-screens
• Scale-up positive screening candidates to a protein midi-preparation using purification machines
• Trouble shoot problems and apply strategies to solve them
• Apply special protein labeling techniques, such as ¹⁵N-labeling for quick NMR analysis
• Crystallize proteins
• Interpret simple NMR measurements
• Carry out electron microscopy sample preparation e.g. negative staining techniques
• Participants will be guided to present short seminar talks of selected topics.
  

Special emphasis for the 2020 course

• Protein expression in eukaryotic cells
  • Expression in Human Embryonic Kidney (HEK) cells (Alexej Schmid)
  • Expression in Baculovirus infected Sf9 cells (Gunter Stier)
  • Expression in plant cell suspensions (Laszlo Bako)

Additional demonstrations and seminars will include the use of:

• Protein purification with “Äkta” and BioRad machines
• Protein stability measurement using Thermofluor assay (Melanie Oelker)
• The Mosquito crystallization robot, crystal imaging facility (Uwe Sauer)
• NMR characterization of ¹⁵N labeled proteins (Anders Öhman)
• EM facility – first approach to electron microscopy measurements (Linda Sandblad)

(*) If you bring your own project(s), please contact the instructors at least 2 weeks before the start of the course, no later than January 15, 2020). It is also possible to work on a project provided by the course leaders.