Samenvatting Eiwittechnologie – Minor Bioresearch 2017 – Joyce Bakker
Samenvatting Eiwittechnologie
Tentamen in het Nederlands, MC en open vragen.
Inhoud
Study task 1: Protein expression. Chapter 6.4-6.7 and Chapter 8.1-8.4 [G&G]....................................................... 3
Explain the phage display technique to identify protein-protein interactions.................................................... 3
Explain the two-hybrid screening technique to identify protein-protein interactions ...................................... 4
Describe the differences between the two techniques phage display and two-hybrid screening and make a
founded choice between the two techniques to identify protein-protein interactions ..................................... 5
Explain the roles of the functional domains of Gal4p in the two-hybrid screening ............................................ 5
Create functional fusion genes in the appropriate vectors for two-hybrid screening ........................................ 6
Explain the problems (and their solutions) with two-hybrid screening .............................................................. 6
Name 4 different reporter genes and their proteins ........................................................................................... 7
Explain the following terms: promoter, ribosomal binding site, multiple cloning site, transcriptional
terminator, selectable marker, origin of replication, Kozak sequence, Shine-Dalgarno sequence, protein over-
production, codon usage, protein tag .................................................................................................................. 8
Appoint the differences between the Shine-Dalgarno sequence and the Kozak consensus sequence .............. 9
Explain the expression systems used in Yeast based on GAL and CUP1 .............................................................. 9
Explain the expression systems used in E.coli based on lac, tac and T7 ............................................................ 10
Explain the mode of action of the Tet-off and the Tet-on systems ................................................................... 11
Study task 2: Protein structures ............................................................................................................................. 13
Describe the 3 methods for protein crystallization ........................................................................................... 13
Explain the physical steps of X-ray scattering by a crystal ................................................................................. 13
Describe the different units of the synchrotron ................................................................................................ 14
Recognize the amino acid side chains R, K, D, E, and C in the context of a polypeptide chain ......................... 15
Study task 3: Enzyme kinetics and Protein purification. Chapter 3, pages 88-110 [ECB], Chapter 36, 43, 46, 47,
54 and 55 [PS+ website 1], Chapter 8.5 [G&G] ...................................................................................................... 16
Describe the terms activation energy, enzyme, substrate, catalyst, free-energy change, delta G, equilibrium
constant K ........................................................................................................................................................... 16
Explain the role of enzymes in chemical reactions using the terms activation energy and delta G ................. 17
Explain the relation between the equilibrium constant K and the concentration of the reactants and products
............................................................................................................................................................................ 20
Give five examples of activated carrier molecules ............................................................................................ 20
Draw the ATP cycle with names, chemical structures and free-energy changes ............................................. 21
Describe the terms v0, Vmax, Km ...................................................................................................................... 22
Reproduce the Michaelis-Menten equation ...................................................................................................... 22
Describe the Lineweaver-Burk plot .................................................................................................................... 22
Explain the principles for isolation using the His- tag, GST-tag, and MBP-tag .................................................. 24
Interpret results from isolations using His- tag, GST-tag, and MBP-tag ............................................................ 26
1
,Samenvatting Eiwittechnologie – Minor Bioresearch 2017 – Joyce Bakker
Make suggestions for problem solving in experimental approaches using His- tag, GST-tag, and MBP-tag .... 27
Calculate the specific activity, purification factor and yield for every purification step ................................... 27
Determine the amount of ammonium sulphate to be added for a particular percentage saturation ............. 27
Describe 4 different differential solubility separation techniques .................................................................... 29
Name the components of a typical homogenization medium .......................................................................... 29
Name 3 examples of non-mechanical disruption methods for cells and tissues .............................................. 29
Name 3 examples of mechanical disruption methods for cells and tissues ...................................................... 29
Explain the roles of the separate steps and the buffer compositions in a given isolation and fractionation
procedure for various organelles ....................................................................................................................... 29
Explain the principles of differential sedimentation .......................................................................................... 30
Explain the principles of density centrifugation ................................................................................................ 30
Calculate the relative centrifugal field (RCF) and rotor speed using equations ................................................ 30
Name differences between types of centrifuge and their uses ......................................................................... 31
Explain the principles of (two-dimensional) SDS-PAGE, iso-electric focusing (IEF) and capillary electrophoresis
............................................................................................................................................................................ 32
Interpret results of (two-dimensional) SDS-PAGE, iso-electric focusing (IEF) and capillary electrophoresis .... 32
Study task 4: Mass Spectrometry. Chapter 42 [PS+ document Mass Spectrometry, website 2] .......................... 33
Explain the principles of Mass Spectrometry (MS) ............................................................................................ 33
Name and draw the 7 main elements of a LC- mass spectrometer ................................................................... 33
Explain the principles of the ionization methods ESI, APCI, APPI ...................................................................... 34
Explain the principles of the mass analysers Quadrupole, Ion Trap and Time-Of-Flight................................... 35
Interpret a given mass spectrum ....................................................................................................................... 37
Study task 5: Chromatography. Chapter 44-45 [PS +, documents Theory of HPLC, website 2] ............................ 39
Name different separation methods.................................................................................................................. 39
Explain the principles of the different separation methods .............................................................................. 40
Name different detection methods ................................................................................................................... 41
Explain the principles of the different detection methods ................................................................................ 42
Calculate the selectivity (α) and the resolution (R) for a column chromatography system .............................. 43
Interpret a given chromatogram ........................................................................................................................ 44
Study task 6: Protein interactions. Document Protein Interactions ...................................................................... 45
Explain the principles of immunoprecipitations, protein-pull down assays and band shift assays .................. 45
Interpret the result of an immunoprecipitation experiment, protein-pull down experiment and band shift
experiment ......................................................................................................................................................... 47
Overige: .............................................................................................................................................................. 49
Proteomics:..................................................................................................................................................... 49
Gel-based analysis .......................................................................................................................................... 49
2
,Samenvatting Eiwittechnologie – Minor Bioresearch 2017 – Joyce Bakker
Study task 1: Protein expression. Chapter 6.4-6.7 and Chapter 8.1-8.4 [G&G]
Explain the phage display technique to identify protein-protein interactions
Techniek voor het identificeren van eiwit interactie met een ander
eiwit. Je weet niet waar bindt of je wilt de beste binder. Identificeren
van peptide of eiwitten die binden met een ander molecuul. DNA met
een specifiek gen of fragmenten worden gekloneerd in een M13 faag,
een virus dat bacteriën infecteert, in frame met gen 3. Het infecteert de bacterie met genetisch materiaal. Het
DNA aan de binnenkant codeert voor de eiwit coat aan de buitenkant. Het gen wordt in het genetisch materiaal
gezet, dit geeft een bepaald eiwit aan de buitenkant. Dus een verband tussen genotype en fenotype.
Panning is een selectieve bindingsstap, alleen fagen
met hoge affiniteit met de binder. Door interactie
verstoren tussen faag en eiwit zullen de binders
loslaten en zullen de M13 fagen met de sequentie met
sterkte binders overblijven. Deze faag samen met
e.coli brengen en dan veel van deze faag verspreiden. M13 plasmiden worden voor veel recombinant
processen gebruikt in vitro mutagenese. Switch tussen enkel- en dubbelstrengs DNA.
Het is dus een techniek voor het identificeren van eiwit-eiwit interacties. Koppeling tussen de bibliotheek van
(willekeurig) eiwit / peptiden (bindend) aan haar gen sequentie. In vitro selectie voor beste bindmiddelen.
Meerdere selectieronden. Fusie van bibliotheek in frame met M13 gen 3, kleine coat PIII. Peptide / eiwit
expressie op het oppervlak van de M13 faag. Gen opeenvolging van bindende peptiden geïsoleerd.
Met behulp van deze techniek kan je de sterkste interactie vinden tussen twee eiwitten. Als je niet weet welke
het beste bindt, met deze techniek laat je alles open. Hiervoor wordt de M13 faag gebruikt (bestaat uit eiwit en
DNA). Infecteert circulair DNA, amplificeert zichzelf in e.coli, het circulair DNA in de faag codeert voor het eiwit
kapsel van de faag. Met behulp van restrictie enzymen wordt tussen de restrictie sites verschillende DNA
sequenties geplakt. Hierdoor heb je talloze verschillende binding sites op de faag. De beste binders binden, de
rest wordt weg gelueerd.
3
,Samenvatting Eiwittechnologie – Minor Bioresearch 2017 – Joyce Bakker
Explain the two-hybrid screening
technique to identify protein-
protein interactions
Een techniek om de interacties tussen
eiwitten te bestuderen in gist cellen.
Door middel van de eucaryotische RNA
polymerase II transcriptionele activatie
eiwitten. Er zijn twee domeinen: een
sequentie specifiek DNA binding
domein (DBD) en een activatie domein
(AD).
Het DBD zorgt voor de binding van de
promoter van specifieke genen, het AD
zorgt voor een site voor complexen die
resulteren in de transcriptie van het gen.
Met de techniek wordt gekeken of twee
specifieke eiwitten met elkaar binden.
Voor deze techniek heb je het volgende
nodig: Rapporteer gen: gen waar je
activatie van kan waarnemen door
middel van bijvoorbeeld fluorescentie.
En een gen transcriptie activator. Om te
testen of de eiwitten interactie met
elkaar hebben zullen de eiwitten
gebonden worden aan AD en DBD. Als
eiwitten binden wordt DNA polymerase
gesignaleerd en het reporter gen
afgeschreven.
Based on transcriptional activation, the yeast Y2H system screens for interacting proteins. The protein of
interest (the bait) is fused to a DNA-binding domain. Proteins that bind to bait (the fish) is fused to a
transcription activation domain. Any protein that binds to the bait will activate the transcription of HIS reporter
gene.
The first step is to construct a bait plasmid and a library of cDNAs in the fish plasmid. Each type of plasmid
contains a selectable marker such as an
essential amino acid. Both types of
plasmids are transformed into yeast
cells. The cells are placed in amino acid
deficient medium, so that only those
containing both plasmids will grow.
Plating on agar lacking histidine selects
for cells that contain the interacting
genes. The binding proteins are
identified by sequencing the DNA’s of
plasmids isolated from these cells.
Transcriptie factor wordt in twee
gesplitst:
4
, Samenvatting Eiwittechnologie – Minor Bioresearch 2017 – Joyce Bakker
DNA Binding Domain (DBD) = bindt aan de promoter van specifieke genen en de Activator domain(AD)
=werving van eiwitten complexen.
Describe the differences between the two techniques phage display and two-hybrid screening
and make a founded choice between the two techniques to identify protein-protein
interactions
Phage display zijn eiwit-eiwit/eiwit-DNA interacties die gebruik maakt van bacteriofagen (virussen die bacteriën
infecteren) om eiwitten te verbinden met de genetische informatie die hen codeert. Bacterie cellen, M13, gen
3 en pIII. Door middel van panning. Voordelen: Identificeerd receptoren, substraten, inhibitoren van enzymen,
epitopen, verbeterd antilichamen en cDNA klonen en Identificeerd peptiden dat meer dan 200x geknipt zijn
efficient.
Two hybrid screening/ Yeast two hybrid screening (Y2H) test de fysische interacties (zoals binding) tussen twee
eiwitten of een eiwit en een DNA-molecuul. Dit door middel van gist cellen (Gal4p). Voordeel: Groot aantal
nummer reporters (cat, lacZ, gusA, luc, GFP).
Explain the roles of the functional
domains of Gal4p in the two-hybrid
screening
Het gist eiwit Gal4p heeft een DNA binding
functie op de amino terminus en een
activatie domein op de carboxy domein. De
GAL4-UAS systeem is een biochemische
methode om genexpressie en functie te bestuderen in
organismen.
Domain structure of Gal4p
DNA Binding Domain (DBD): aa 1-65
Dimerization Domain: aa 65-94
Transcription activation: aa 148-196
TransActivation Domain (AD): aa 768-881
Gal80p binding site (transcriptie repressor): aa 851-881
DNA Binding Domain (DBD): aa 1-65 of Gal4p
• Binds as a dimer
• Zn(II)2Cys6 binulear cluster
• Two zinc ions
• Upstream Activation Squence voor galactose (UASG):
5’-CGGN11CCG-3’
5
