NANOBODIES: promising reagents in basic¨кола'2017... · 2018-09-09 · NANOBODIES: promising...
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NANOBODIES: promising reagents in basic research and biomedicine Introduction Antibodies (Abs) are proteins produced by the immune system to capture antigen epitopes . There are two types of antibodies: conventional (eg IgG) and heavy chain Abs (HCAb). Conventional antibodies consist of heavy and light chains and interact with antigens via both variable domains. HCAb is a type of antibody comprising only of heavy-chain domains. They were found in 1990’s in the family Camilidiae and cartilaginous fishes. The VHH domain on the HCAb is referred to as the nanobody. Антитела - это белки, вырабатываемые иммунной системой для захвата антигенов. Существует два типа антител: 1 - состоящие из тяжелых и легких цепей , 2 – состоящие только из тяжелых цепей (HCAb). HCAb были обнаружены у сем. Верблюдовые и у хрящевых рыб в 1990-х. VHH домен HCAb называют наноантителами. Conventional antibodies vs Heavy chain antibodies. Jane Nekrasova, Julia Urgel, Katya Shuvalova, Natalia Ketaren, Methods Project 2: Varying PEG types and concentrations for capturing Nup84 complex Antibodies can be monoclonal, where they are specific only for one epitope on an antigen and polyclonal, where there are mixtures of antibodies that can bind an antigen on many different epitopes. The main aim of this project is to estimate whether monoclonal or polyclonal antibodies allows us to capture our antigen (Nup84). Thus, we proceed to produce mixtures of individual nanobodies and compare their efficiency in affinity capture experiments to monoclonal antibodies. RESULTS Protein-protein interactions laboratory (LaCava Lab) Conventional antibody Heavy-chain antibody Will denature at higher temperature and pH Resistant to heat and pH Large Small in size Has two HC and two LC Has variable HC domain Complex protein Simple protein outsourced Can make in lab Nup84 complex The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). This complex is composed of 7 different proteins. The main functions of Nup84 complex are related to nucleocytoplasmic transport and NPC biogenesis. It also plays roles in several processes that may require localization of genes or chromosomes at the nuclear periphery, including double- strand break repair, transcription and chromatin silencing. Комплекс Nup84 представляет собой ключевой строительный блок в ядерном поровом комплексе (NPC). Этот комплекс состоит из 7 различных белков. Основные функции Nup84 - это осуществление нуклеоцитоплазматического транспорта и формирование NPC. Он также участвует в некоторых процессах, необходимых для локализации генов или хромосом в ядре, включая репарацию двунитевых разрывов, транскрипцию и сайленсинг хроматина. Fernandez-Martinez et al.; 2012 MW of Nups in Nup84 complex - Nup133 = 129 kDa - Nup120 = 120 kDa - Nup84+GFP = ~112 kDa - Nup145c = ~82 kDa - Nup85 = ~80 kDa - Seh1 = 39 kDa - Sec13 = 33 kDa Dynabeads conjugation with nanobodies 0.1 M Sodium phosphate 1 M Ammoniumsulphate Nanobodies 37 ℃ overnight Wash beads Dynabeads are paramagnetic beads that carry an epoxy group. These epoxy groups covalently bind to amine groups on the proteins. Project 1: Monoclonal and polyclonal nanobodies for capturing Nup84 complex Affinity capture experiments: DISCUSSION & CONCLUSIONS Method 1: mixing different nanobodies, then conjugating mixture to beads Method 2: mixing different beads conjugated with single nanobodies Western-blot experiments SDS-PAGE electrophoresis gel In order to separate proteins according to their molecular weight we used SDS-PAGE. Bicinchoninic acid (BCA) assay Protein Cu 2+ + Temperature dependent reduction Cu + OH - + 2 Cu + is proportional to the amount of protein Cu + +BCA 2 is proportional to the amount of protein Abs562 nm is proportional to the amount of protein HRP Primary antibody Secondary antibody Target Protein (GFP) Target protein containing GFP binds to anti-GPF primary antibody. Secondary antibody with HRP allows us to visualize protein bands. mPEG-SCM (PEG-NHS: Succinimidyl Carboxyl Methyl ester) Pegylation reaction Polyethylene glycol (PEG) is a polymer that is commonly used to assist potential drug targets being recognized by the immune system. The purpose of this project is to optimize the nanobody-pegylation reaction by varying different parameters in the reaction mixture. Parameters we aim at changing are (i) concentration of nanobody; (ii) concentration of PEG reagent and (iii) incubation time. We will perform the reactions using two PEGs with two different molecular weights, being 5 kDa and 20 kDa. Monoclonal Vs Polyclonal affinity capture (AC) RESULTS LOADING LIST 1. MW Marker 2. BSA 20 ug/mL 3. LaG 1 4. LaG 2 5. LaG 3 6. LaG 4 7. LaG m18 8. LaG 1+ LaG 4 9. Lag 1+ JaG m18 10. LaG 2+ LaG 3+ LaG 4 11. LaG 2+ LaG 3+ LaG m18 12. LaG m18+ LaG 2 13. LaG 3+LaG 2 14. LaG 3+ LaG m18 15. Control LaG 1 MONOCLONAL POLYCLONAL (kDa) 250 148 98 64 50 36 22 16 6 4 Figure 2 (A)- SDS-PAGE Blue Silver R250 staining and (B) corresponding Western Blot with α-GFP antibody Figure 1 SDS-PAGE gel of different affinity capture experiments comparing monoclonal nanobodies to polyclonal nanobodies. DISCUSSION AND CONCLUSIONS Figure 3 SDS-PAGE protein gel showing results of varying different parameters in the pegylation reaction From the SDS-PAGE of affinity capture experiments we cannot definitively identify the protein Nup84-GFP and its associated proteins for a few reasons: - Due to unusual molecular weight standard mobility compared to BSA control - Due to the poor resolution in the AC experiments. Thus we performed a Western blot to identify target Nup84-GFP protein localization using anti-GFP antibody. The results allowed us to determine that Nup84-GFP was indeed captured. We observed that the bands differ in intensity and that most of the polyclonal combinations resulted in a higher intensity band. Thus we can interpret a highest affinity when mixing monoclonal nanobodies in order to obtain polyclonal nanobodies. The most effective combinations results from LaG 3 and m18. This combination performs the same affinity of the control. Nevertheless, we can underline that m18 by it self does not show any band. From the SDS-PAGE in Figure 3, we see that increasing the concentration of m18 whilst keeping the concentration of PEG constant, higher concentrations of m18 in the reaction shows more un-pegylated m18. We see that the reactions with 20 kDa PEG did not modify as many lysines as 5 kDa PEG. Wee see that decreasing the concentration of 5 kDa PEG results in less modified lysines. Because of this, we conclude that a larger ration of [PEG]:[protein] is necessary to favor more pegylated lysines.
Transcript of NANOBODIES: promising reagents in basic¨кола'2017... · 2018-09-09 · NANOBODIES: promising...
NANOBODIES: promising reagents in basic research and biomedicine
Introduction
Clonalrelationshipandcellbehaviorofneuronalprogenitorsinthehindbrain
Antibodies (Abs) are proteins produced by the immune system to capture antigen epitopes.There are two types of antibodies: conventional (eg IgG) and heavy chain Abs (HCAb).Conventional antibodies consist of heavy and light chains and interact with antigens via bothvariable domains. HCAb is a type of antibody comprising only of heavy-chain domains. They werefound in 1990’s in the family Camilidiae and cartilaginous fishes. The VHH domain on the HCAb isreferred to as the nanobody.Антитела - это белки, вырабатываемые иммунной системой для захвата антигенов.Существует два типа антител: 1 - состоящие из тяжелых и легких цепей , 2 – состоящиетолько из тяжелых цепей (HCAb). HCAb были обнаружены у сем. Верблюдовые и у хрящевыхрыб в 1990-х. VHH домен HCAb называют наноантителами.
Conventional antibodies vs Heavy chain antibodies.
Jane Nekrasova, Julia Urgel, Katya Shuvalova, Natalia Ketaren,
Methods
Project 2: Varying PEG types and concentrations for capturing Nup84 complex
Antibodies can bemonoclonal, where they are specific only for one epitope on an antigen and polyclonal, wherethere are mixtures of antibodies that can bind an antigen on many different epitopes.The main aim of this project is to estimate whether monoclonal or polyclonal antibodies allows us to capture ourantigen (Nup84). Thus, we proceed to produce mixtures of individual nanobodies and compare their efficiency inaffinity capture experiments to monoclonal antibodies.
RESULTS
Protein-protein interactions laboratory(LaCava Lab)
Conventional antibody Heavy-chain antibodyWill denature athighertemperature andpH
Resistant toheat andpH
Large SmallinsizeHastwo HCandtwo LC HasvariableHCdomain
Complex protein Simpleproteinoutsourced Canmake inlab
Nup84 complexThe Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). This complex is composed of 7 differentproteins. The main functions of Nup84 complex are related to nucleocytoplasmic transport and NPC biogenesis. It also playsroles in several processes that may require localization of genes or chromosomes at the nuclear periphery, including double-strand break repair, transcription and chromatin silencing.Комплекс Nup84 представляет собой ключевой строительный блок в ядерном поровом комплексе (NPC). Этот комплекссостоит из 7 различных белков. Основные функции Nup84 - это осуществление нуклеоцитоплазматического транспортаи формирование NPC. Он также участвует в некоторых процессах, необходимых для локализации генов или хромосом вядре, включая репарацию двунитевых разрывов, транскрипцию и сайленсинг хроматина.
Fernandez-Martinez etal.;2012
MWofNups inNup84complex- Nup133=129kDa- Nup120=120kDa- Nup84+GFP=~112kDa- Nup145c=~82kDa- Nup85=~80kDa- Seh1=39kDa- Sec13=33kDa
Dynabeads conjugation with nanobodies
0.1MSodiumphosphate
1MAmmoniumsulphate
Nanobodies37℃
overnightWashbeads
Dynabeads are paramagnetic beads that carry anepoxy group. These epoxy groups covalently bind toamine groups on the proteins.
Project 1: Monoclonal and polyclonal nanobodies for capturing Nup84 complex
Affinity capture experiments:
DISCUSSION & CONCLUSIONS
Method 1: mixing different nanobodies, thenconjugating mixture to beads
Method 2: mixing different beads conjugated withsingle nanobodies
Western-blot experimentsSDS-PAGE electrophoresis gel
In order to separate proteinsaccording to their molecular weightwe used SDS-PAGE.
Bicinchoninic acid (BCA) assay
ProteinCu2+
+
Temperature dependentreduction
Cu+OH-
+ 2
Cu+ isproportionaltotheamountofproteinCu++BCA2 isproportionaltotheamountofproteinAbs562nmisproportionaltotheamountofprotein
HRP
Primaryantibody
Secondaryantibody
TargetProtein(GFP)
Target protein containing GFP bindsto anti-GPF primary antibody.Secondary antibody with HRP allowsus to visualize protein bands.
mPEG-SCM(PEG-NHS:SuccinimidylCarboxylMethylester)
Pegylation reaction
Polyethylene glycol (PEG) is a polymer that is commonly used to assistpotential drug targets being recognized by the immune system.The purpose of this project is to optimize the nanobody-pegylationreaction by varying different parameters in the reaction mixture.Parameters we aim at changing are (i) concentration of nanobody; (ii)concentration of PEG reagent and (iii) incubation time. We will performthe reactions using two PEGs with two different molecular weights, being5 kDa and 20 kDa.
Monoclonal Vs Polyclonal affinity capture (AC)
RESULTS
LOADINGLIST1. MWMarker2. BSA20ug/mL3. LaG 14. LaG 25. LaG 36. LaG 47. LaG m188. LaG 1+LaG 49. Lag1+JaG m1810. LaG 2+LaG 3+LaG 411. LaG 2+LaG 3+LaG m1812. LaG m18+LaG213. LaG 3+LaG214. LaG 3+LaG m1815. ControlLaG 1
MONOCLONAL POLYCLONAL
(kDa)250148
9864
50
36
221664
Figure 2 (A)- SDS-PAGE Blue Silver R250 staining and (B) correspondingWestern Blotwith α-GFP antibody
Figure 1 SDS-PAGE gel of different affinity capture experiments comparing monoclonalnanobodies to polyclonal nanobodies.
DISCUSSION AND CONCLUSIONS
Figure3SDS-PAGEproteingelshowingresultsofvaryingdifferentparametersinthepegylationreaction
From the SDS-PAGE of affinity capture experimentswe cannot definitively identify the protein Nup84-GFPand its associated proteins for a few reasons:- Due to unusual molecular weight standard
mobility compared to BSA control- Due to the poor resolution in the AC experiments.Thus we performed a Western blot to identify targetNup84-GFP protein localization using anti-GFPantibody. The results allowed us to determine thatNup84-GFP was indeed captured.We observed that the bands differ in intensity andthat most of the polyclonal combinations resulted in ahigher intensity band. Thus we can interpret a highestaffinity when mixing monoclonal nanobodies in orderto obtain polyclonal nanobodies. The most effectivecombinations results from LaG 3 and m18. Thiscombination performs the same affinity of thecontrol. Nevertheless, we can underline that m18 by itself does not show any band.
From the SDS-PAGE in Figure 3, we see thatincreasing the concentration of m18 whilstkeeping the concentration of PEG constant,higher concentrations of m18 in thereaction shows more un-pegylated m18.We see that the reactions with 20 kDa PEGdid not modify as many lysines as 5 kDaPEG.Wee see that decreasing the concentrationof 5 kDa PEG results in less modifiedlysines.Because of this, we conclude that a largerration of [PEG]:[protein] is necessary tofavor more pegylated lysines.
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