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Course Examination



In order to pass the exam, you need to solve 6 (out of 8) tasks (but solving task 1 is obligatory) and answer to 7 (out of 9) questions. Only correct answers will be counted! You will also get 2 points for each correctly solved task and 1 point for each correct answer. Hence, you will be able to get maximum 25 points, which will be added to 40 points awarded for the accomplishment of labs (plus 1-5 points given by lab teaches for good performance), 0-10 points given for the VNTI lab exercises, 0-10 for the “scientific report”, 0-5 points given for the oral report on fungal expression lab, and 0-5 points given for the presentation of a purification method. Although this is a “home exam”, you should perform it individually! Please, send your answers to Anton no later than the 15th of January.


Jan 12th, Thursday

Work on the exam tasks.


You can do the exam where you like, but note that from 9:00 to 15:00 Datorsal 2 in the library is reserved entirely for you.



Jan 13th, Friday

Work on the exam tasks


You can do the exam where you like, but note that from 9:00 to 15:00 BU27 (Datorsal) is reserved entirely for you (VNTI is not installed here!).


Task 1


Using VNTI software, virtually clone the target gene (see sequence bellow) into the expression pET101/D-TOPO vector (see exercise 1 of the Design of expression constructs lab). Draw the scheme of the resulting plasmids. Design PCR primers for target gene amplification and cloning. Report (1) scheme of the plasmid, (2) sequence of the plasmid with translated target gene, and (3) PCR primers.


Sequence of the gene:


Group 1: Maruf, Elham,


Group 2: Wenbo, Kibria


Group 3: Mahafuzur, Mehrafarin


Group 4: Umair, Shirin


Group 5: Yagmur, Kumer


Task 2


Suppose you managed to express your target protein (see Task 1) in the cytoplasm of E. coli and the protein accumulated in the fraction of soluble proteins. Suggest a possible purification scheme for this protein. Start with the following sentence:

“Cells will be harvested by centrifugation and …”


Task 3


You want to purify your target protein (see Task 1) with ion-exchange. At which pH will you purify the protein on (1) MonoQ (positively charged groups) and (2) MonoS (negatively charged groups) columns? Which buffers will you use for the purification? Explain how you will prepare 1 liter of binding and elution buffers of your choice (for MonoS only). Calculate the weights of reagents that you will use to prepare the buffers.


Hints: Determine pI of your protein using VNTI (create protein molecule), check the molecular weight of reagents on the bottles in our chemical storage


Task 4


You have purified your target protein (see Task 1). The sample of you protein showed absorbance of 2.5 units at 280 nm on our nanodrop spectrophotometer. Estimate protein concentration in g/l and in mol/l.


Hints: Determine molecular weight and absorbance of 1 mg/ml solution of your protein using VNTI (create protein molecule)


Task 5


You have determined dissociation constant for the binding FimH-LD to mannose. What was its value? Suppose you mixed 0.5 ml 5 mM FimH-LD and 0.5 ml mM 5 mM mannose. What will be the concentration of the FimH-LD-mannose complex in the mixture (concentration at equilibrium)? Solve this task either explicitly or by substitution.


Hints: to solve the task explicitly, remember how to solve equations of the following type: ax2+bx+c=0 (quadratic equation)



Task 6


Many proteins have TIM-like (b/a)8 barrel and immunoglobulin-like folds. Find two proteins, which have these folds, and create a protein molecule in VNTI software for each of these proteins. Annotate the molecules (e. g. mark secondary structure elements, b strands of beta sheets and a helices). Calculate the fraction of hydrophobic residues in b strands of the TIM-like (b/a)8 barrel and immunoglobulin-like proteins. You suppose to get two significantly different values. Can you explain the result? What are the functions of your proteins?


Hints: Read about the folds and draw topology diagrams


Task 7


Draw a schematic diagram of how cholesterol is incorporated into the lipid bilayer showing the correct orientation and explain how does the cholesterol molecule affect physical properties of membrane.


Hints:  Look at this picture


Task 8


Very often the term “unit” is used as a measure of amount of enzyme available. How would you define a unit?


The results shown in the table (click here) were obtained during the isolation of a

 protein. The measurements shown in the table were done after each step, for

 example 40 ml of the protein solution was obtained after AS-P step.


1. Calculate the specific activity and fold purification of the protein for each step and fill in the table.


2. Which step did not increase the purity of the protein? Why may this step nevertheless be important in this purification?


3. How could you obtain additional evidence on the purity of this protein?





Q 1

Which methods of biophysical characterizations of enzymes do you know? Explain briefly each method.


Q 2

Protein purification techniques are based on several properties of protein of the interest in solution. In the list below, several techniques are mentioned. State what property of the protein is important for each technique.


· Affinity chromatography

· Ammonium sulfate precipitation

· SDS gel electrophoresis

· Isoelectric focusing

· Sedimentation

· Ion exchange chromatography

· Size exclusion chromatography


Q 3

Which expression hosts would you choose for protein expression if


(1) The target protein is from a bacterium; you need a small quantity of this protein (1-2 mg) for scientific purposes

(2) The target protein is human insulin; you need to produce 600 kg of this protein (a typical annual consumption of a large country like USA) (you can use internet to find the answer)?


Explain the choice of the expression host.


Q 4

Write down the equations describing (1) saturation curve in saturation binding experiment and (2) inhibition curve in inhibition binding experiment (we assume that concentration of ligands in the experiment is much higher than that of the receptors).


Q 5

Explain the basic principles of association and dissociation rate constants determination using Biacore instrument. Binding of proteins A and B is characterized with association and dissociation rate constants 20000 (s-1 M-1) and 0.01 (s-1), respectively, whereas binding of proteins C and D is characterized with association and dissociation rate constants 40000 (s-1 M-1) and 0.04 (s-1), respectively. Which binding has higher affinity, A-B or C-D?


Q 6

What is

(1) lectin protein

(2) molecular chaperone

(3) adhesive pili or fimbriae


Q 7

What expression levels of a target protein (in mg) can one maximum expect to get if he/she is using an expression host like:


     1. Hypocrea jecorina

     2. Pichia pastoris

     3. E. coli

     4. Insect cell lines transformed using Baculovirus


Describe in general terms some of the advantages/disadvantages using the four above mentioned expression systems


Q 8

Describe why one need to include antibiotica in the growth medium when expressing a target protein in E. coli, and why this (in most cases) is not needed when expressing a protein in a eukaryotic expression system.


Q 9

(a) List hydrophobic, polar, and charged at pH 7-7.5 side chains of amino acid residues. (b) Side chains of which residues typically have pKa values close to 7? (c) Is it possible for a protein to have pI value of 1 (pI=1)? (d) Explain the hydrophobic effect (hydrophobic interactions).






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