BMB523: Recombinant human protein kinases: expression, purification, characterization (5 ECTS)

STADS: 01004601

Level
Bachelor course

Teaching period
The course is offered in the spring semester.
3rd quarter.

Teacher responsible
Email: ogi@bmb.sdu.dk

Additional teachers
bag@bmb.sdu.dk

Timetable
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Comment:
Maksimalt deltagerantal 16

Prerequisites:
None

Academic preconditions:
BMB504 and BMB503 must have been followed.

Course introduction
Using the expression vector pMal an MBP-tagged protein kinase is expressed in E. coli. Bacterial pellets containing the recombinant MBP-tagged protein kinase are used to prepare a lysate. The enzyme is purified and characterized. Techniques: Radioactive protein kinase activity test. Affinity chromatography on amylose. SDS-PAGE analysis. Westernblot/immunodetection. Proteolytic cleavage of the MBP tag. Stability investigations with and without MBP-tag. In vitro assembly of kinase subunits. Test for tetramer formation by gel filtration (SMART). Most of the kinases require phosphorylation for their activity. Using a luminometric test, check the activity status of protein kinases before and after treatment with phosphatase. Expression and autoradiographic detection of a kinase produced in a coupled transcription/translation assay (RTS, Roche).

Qualifications
Explain how expression plasmids are designed.
nt protein tags incl. affinity chromatography.
Proteolytic cleavage
Handling of radioactive isotopes
Perform protein kinase tests and kinetic calculations
Explain how protein kinases are regulated
Know the background for coupled transcription/translation expression systems

Expected learning outcome
At the end of the course, the student is expected to be able to explain at the solo 5 level:

Design a strategy for the cloning of cDNA into bacterial expression plasmids
Describe how a successful cloning can be verified, and how expression of recombinant protein can be checked.
Describe different affinity chromatography techniques to purify tagged recombinant proteins
Describe how to perform a proteolytic cleavage experiment to remove the tags from recombinant proteins
Handling of radioactive isotopes safely and correct (describe the safety procedures which have to be followed while handling radioactive isotopes)
Describe how to perform protein kinase tests, incl. the calculation of kinetic parameters
Explain the basic principle of gel filtration analyses
Explain the role of phosphatases in signal transduction and how dephosphorylation reactions of active protein kinases is performed.
Explain the role and importance of specific phosphase inhibitors
Describe how to perform a luminometer-based protein kinase test with active enzymes (phosphorylated) and unactive kinases (not phosphorylated).
Describe the chemical basis for a luminometric test
Describe the background for a coupled transcription/translation assay and how to perform the test

Subject overview
Accompanying lectures cover the major topics such as for example: expression systems, use of different tags, affinity chromatography, characterization of recombinant proteins, gel filtration, activity tests(radioactivity, luminometry). Coupled in vitro transcription /translation for the production of recombinant proteins.

Literature
There isn't any litterature for the course at the moment.

Syllabus
See syllabus.

Website
This course uses e-learn (blackboard).

Prerequisites for participating in the exam
None

Assessment and marking:
Written report
The report is written in groups of two people and is internally evaluated by the responsible teacher as passed/not passed.

Expected working hours
The teaching method is based on three phase model.

Forelæsninger, antal timer 5.
Laboratorieøvelser, antal timer 45.

Deltagerne må holde små oplæg, der forklarer teorien bag eksperimenterne (optionelt).
Educational activities

Language
This course is taught in English, if international students participate. Otherwise the course is taught in Danish.

Course enrollment
See deadline of enrolment.

Tuition fees for single courses
See fees for single courses.