Proteins and nucleic acids

elective courses

Biochemistry 1400-113BCH
Molecular Biology 1400-215BM

Biochemistry 1400-113BCH
Molecular Biology 1400-215BM

Student should know the principles of biochemistry of proteins and nucleic acids as well as basic techniques like electrophoresis and chromatography. Knowledge presented on “Biochemistry” course is sufficient to be qualified for the course.

Classroom

The objective of the course is to present methods used to study proteins. Students purify proteins from natural sources using conventional protocols and they learn novel isolation techniques used for purification of recombinant proteins.

Laboratory session topics include:

1. Purification of lysosyme from white egg. The protocol includes conventional multistep procedure used for protein purification (choice of biological material, isolation of the fraction enriched in purified protein, differential precipitation, dialysis, ion exchange chromatography, size exclusion chromatography, gel electrophoresis).

2. Purification of the recombinant human splicing factor SRSF1 from Escherichia coli cells. Affinity gels are used which allow protein purification with a one-step procedure. Short polyhistidine tag (6xHis) or maltose binding protein (MBP) are added to the purified protein by genetic engineering techniques. The recombinant proteins bind to affinity resins that contain immobilized nickel or maltose, respectively. This is an example of a micropreparative procedure.

3. Preparation of HeLa nuclear extract and determination of the enzymatic activity of topoisomerase I. The example of simple fractionation method on small scale preparation from human cell culture. This practice also shows the method of examination of relaxation activity of topoisomerase I. The result of reaction – partially and fully relaxed forms of plasmid are resolved with agarose gel electrophoresis.

4. Independent analysis of scientific publications, construction of a method for purifying proteins from natural sources, and evaluation of the effectiveness of the proposed purification procedure. The exercise consists of two elements: planning your own method for purifying a specific protein from natural sources, and purifying the protein while assessing the quality of the obtained preparation. Through this exercise, participants will gain the ability to independently construct a purification method for selected proteins.

Hames, B.D. and Hooper, N.M. (2000) Instant Notes in Biochemistry. Bios Scientific Publishers Ltd.

Kłyszejko-Stefanowicz, L., red. (1999) Ćwiczenia z biochemii (in Polish). PWN, Warszawa.

Anonymous author (1998) Protein purification handbook. Amersham Pharmacia Biotech, Uppsala. (to be found in Dept. of Molecular Biology or www.apbiotech.com)

The student is required to complete four exercises described in the "full description" section. After conducting each exercise, the student should present the results to the course instructor, summarize the course of the exercise, and interpret the results. The instructor may ask additional questions, but they should not go beyond the scope of the discussed exercise. Correctly addressing the above points allows for the completion of the exercise. Passing all exercises is a condition for being eligible to participate in the final presentation (on exam terms).

During the final presentation, participants present selected issues related to the exercise content or their own results from laboratory procedures conducted during the classes in written form (report or poster) or orally. The presentation is evaluated by the course instructors based on the completeness of the information presented and the substantive value.

Not required.