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Carbohydrate, isolation and purification techniques. A complete view.

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Carbohydrate, isolation and purification techniques. A complete view.

  1. 1. Basic Techniques Used in Molecular Biology For The Cloning, Expression, Purification and Characterisation of a Family 8 Glycoside Hydrolase from Clostridium thermocellum By Neha Mishra Under supervision of Dr. Arun Goyal (IITG) Biochemical Engineering (3rd year) Bipin Chandra Tripathi Kumaun Institute of Technology Dwarahat (Almora 263653), UKD (2016-2017) 1
  2. 2. The CAZy database CAZy is a database of Carbohydrate-Active enzymes (CAZymes). The database contains a classification and associated information about enzymes involved in the synthesis, metabolism and transport of carbohydrates. Included in the database are glycoside hydrolases, glycosyltransferases, polysaccharide lyases , carbohydrate esterase and carbohydrate-binding families. 2
  3. 3. Family 8 Glycoside Hydrolase from Clostridium thermocellum(CtGH8) • Glycoside hydrolases are a group of enzymes which catalyse the hydrolysis of the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety. • CtGH8 catalyses the hydrolysis of β-1,4-glycosidic bonds present in cellulase, chitosan and xylan. 3
  4. 4. Polymerase chain reaction Polymerase Chain Reaction (PCR) is a molecular biology technique which is used to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing. There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. 1. Denaturation at 94°C 2. Annealing at 50°C-54°C 3. Extension at 68°C-72°C 4
  5. 5. s Optimized PCR conditions for family 8 glycoside hydrolase from Clostridium thermocellum PCR components volume used(µl) Final concentration 10x reaction buffer 5.0 1x 10mM dNTPs 3.0 1mM Forward primer 1.5 0.5µM Reverse primer 1.5 0.5µM Genomic DNA (2.26ng/ µl) 1.0 picogram/µl Taq DNA polymerase (NEB) 1.5 0.025U/µl Sigma water 36.5 Total 50.0 Steps Time (minute) • Denaturation at 94 °C 4:00 • 30 cycles of 1. Denaturation at 94°C 0:30 2. Annealing at 64.5°C 0:45 3. Extension at 68°C 1:00 • Final extension at 68 °C 10:00 Fig 1: Agarose gel (0.8%) showing PCR amplified fragment of CtGH8 Lane 1: DNA marker, Lane 2: Amplified CtGH8 at 64.5°C. 5 kb
  6. 6. 1. Harvest & lyse bacterial cells . 2. Pellet cells from 1-5ml overnight culture, discard supernatant. 3. Resuspend cells in 200l resuspention solution. Pipette up & down/vortex. 4. Add 200l of lysis solution. Invert gently to mix. Allow to clear for 5 min (Prior to first time use, be sure to add RNase A to the resuspension solution) 5. Add 350l of neutralization solution to the prepared cleared lysate. 6. To prepare binding column add 500ml column preparation solution to a binding column in a collection tube. Spin and discard flow through. 7. Next transfer cleared lysate into binding column. Spin and discard flow through. 8. Add 750l wash solution to column. Spin and discard flow-through. 9. Spin 1 minute to dry column. 10. Transfer column to new collection tube. Add 100l elution solution to elute the plasmid DNAs. Bacterial culture Pure plasmid DNA Isolation of plasmid pET28a(+) DNAs using GenEluteTM Plasmid Miniprep Kit 6
  7. 7. kb 10 6 3 2 1.5 1 2 1 0.5 5.369kb Fig 2: 0.8% Agarose gel. Lane1: Plasmid DNA, Lane2: DNA marker 7
  8. 8. Agarose gel electrophoresis  Agarose is a polysaccharide extracted from seaweed.  Easy to prepare.  Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix.  The biomolecules are separated on the basis of size. The pore size of agarose gel is 50 nm >200nm.  Agarose gels have a large range of separation by varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:  An electrophoresis chamber and power supply.  Gel casting trays; available in a variety of sizes and composed of UVtransparent plastic.  Sample combs; around which molten agarose is poured to form sample wells in the gel.  Electrophoresis buffer; usually Trisacetate EDTA (TAE) or Trisborate EDTA (TBE).  Loading buffer; contain something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.  Ethidium bromide; fluorescent dye used for staining nucleic acids.  Trans illuminator (an ultraviolet lightbox); which is used to visualize ethidium bromide stained DNA in gels. 8
  9. 9. Fig 3: Agarose gel 0.8% showing, Lane1: DNA marker (NEB), Lane 2: Restriction Digestion of plasmid containing CtGH8, showing pET28a(+) vector (5.34Kb) & CtGH8 (1.059Kb) 9 kb
  10. 10. Escherichia coli BL 21 Competent cell preparation The charges of Ca2+ neutralize the negative charge of both the plasmid and bacterial cell wall, dissipating the electrostatic repulsion and weakening of cell wall. Procedure;  Revive E.coli BL21 (from glycerol stock) in 5ml LB and incubate at 37C for (12-16 hour) at 180 rpm.  Incubate the 0.25ml of the growth culture into 25ml of Luria-Bertani(LB) and incubate at 37c till it reach the OD 0.3-0.4 at 600nm.  When suitable growth has been reached chill the culture on ice for 15 minutes.  Centrifuge at 2710×g for 10 min at 4C, discard the supernatant.  Suspend the pellet with 0.1M CaCl2 (5ml) and incubate in ice for 10 minutes.  Again centrifuge for 10 minutes.  Resuspend the cells in 0.1M MgCl2 (5ml) and incubate on ice for 15 minutes.  Centrifuge it discard supernatant and suspend it in 1ml of 0.1M CaCl2 containing 15% glycerol (ice cold).  Store it in -80 C. 10
  11. 11. Bacterial Transformation by Heat Shock method 1. Thaw 100 l of competent cells in ice. 2. Add 1 l of plasmid to 100 l of competent cell 3. Incubate the cells on ice for 30 minutes 4. Quickly transfer in water bath at 42C for 40 second, transfer on ice for 5 minutes 5. Add 900 l of LB and incubate at 37C for 1 hour, centrifuge at 5,000g for 30 minutes and discard the supernatant. Then dissolve the pellet in remaining 100 l of medium 6. Spread out this culture on to the plates containing appropriate antibiotic Expression of CtGH8 in BL-21 Cells  Inoculate 1.0ml of cells in 100ml LB media + 50µl kanamycin (50µg/ml) and incubate at 37C, 180 rpm.  When OD reaches to 0.6 at 600nm then induce with Isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubate at 24C, 180rpm for 16-18 hours.  The expression of recombinant proteins was checked by loading both uninduced and induced cell samples on SDS-PAGE (12%). 11
  12. 12. Expression of CtGH8 Fig 4:SDS- PAGE (12%) gel showing expression of recombinant CtGH8 in E.coli BL-21 cells after induction with IPTG. Lane 1: uninduced cells, Lane 2: induced cells 1 at 37°C, Lane 3: induced cells 2 at 37°C, Lane 4: induced cells 3 at 37°C, Lane 5: induced cells 1 at 24°C, Lane 6: induced cells 2 at 24°C, Lane 7: induced cells 3 at 24°C, Lane 8: induced cells 4 at 24°C, M: Protein marker. 12 kDa 39kDa
  13. 13. SONICATION  Centrifuge the culture at 8000 rpm for 10 minutes at 4C.  Cell pellet obtained, dissolve in lysis buffer, 6 ml lysis buffer for 100 ml culture.  Add 10 µl of (1mM) PMSF (it is a general serine protease inhibitor. It is the most common inhibitor used in protein purification, soluble in isopropanol and may be stored at -20ºC for a year), mix.  Sonicate 30 minutes(9sec on, 15sec off).  Till culture becomes transparent.  Centrifuge 13,000 rpm for 15 minutes. Buffers used in protein purification 1.Binding buffer : 50mM TrisHCl, 300mM NaCl, 50mM Imidazole, pH 7.5 2.Elution buffer : 50mM TrisHCl, 300mM NaCl, 300mM Imidazole, pH 7.5 3.Dialysis buffer: 50mM TrisHCl, 300mM NaCl, pH 7.5 13
  14. 14.  3/5 ml of 0.1M Nickel sulphate, incubate for 15 minute.  Wash with degassed H2O 5 volume of column.  Pass binding and washing buffer 10ml Add sample supernatant  Wash with binding/washing buffer (20 times).  Collect last column fraction, keep it in fridge.  Elute the protein using elution buffer 5ml.  Check the protein concentration using bradford method. Purification of CtGH8 by 1ml GE Gravity column  Take the used column now and wash with degased miliQ water 20 ml.  Wash with cleaning buffer (fridge 10ml).  Wash with miliQ H2O (20-40ml).  Pass 6ml 1N NaOH, incubate for 2 hours.  Wash with degassed miliQ water 50-100ml.  Fill the column with 20% ethanol and store it in fridge for further use. Cleaning of column 14
  15. 15. Fig 5:SDS-PAGE (10%) gel showing expression and purification of recombinant CtGH8 in E. coli BL-21 cells. Lane 1: uninduced cells, Lane 2: induced cells, Lane 3: cell pellet after sonication, Lane 4: cell free extract, Lane 5: last wash from column, Lane 6: column purified protein, M : protein marker. 15 kDa 39 kDa
  16. 16. PAGE (Polyacrylamide gel electrophoresis)  To separate and characterize proteins.  Here a solution of acrylamide and bisacrylamide is polymerized.  Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS).  Tetramethylethylenediamine (TEMED), a free radical stabilizer, is generally included to promote polymerization. SDS PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis)  Sodium dodecyl sulfate (SDS) is an amphipathic detergent. It has an anionic head group and a lipophilic tail. It binds noncovalently to proteins.  It also confers negative charge. The process basically includes three steps 1. Preparation of the Gel 2. Sample Preparation and loading of samples 3. Electrophoresis Principle of SDS PAGE 16
  17. 17. SDS-PAGE gel and buffer composition Components VT= 5ml VT= 10ml dH2O 2.8ml 5.6ml 30% acrylamide 0.7ml 1.4ml 10% SDS 0.5ml 1.0ml Tris (6.8) pH 1.0ml 2.0ml 10% APS 50l 100l TEMED 5l 10l 4% SDS-PAGE stacking gel Components VT=10ml VT=12ml VT=14ml dH2O 1.4ml 0.7ml 0.03ml 30% acrylamide 3.3ml 4.0ml 4.67ml 10% SDS 1.0ml 1.0ml 1.0ml 50% glycerol 1.0ml 1.0ml 1.0ml Tris HCl (pH 8.8) 3.3ml 3.3ml 3.3ml 10% APS 100l 100l 100l TEMED 10l 10l 10l Resolving gel composition 17
  18. 18. Composition of Tris-Glycine running buffer or tank buffer Composition of 5X sample loading buffer Components Final concentration Tris HCl (pH 6.8) 62.5mM Glycerol 20(%w/v) SDS 2.0(%w/v) Bromophenol 0.025(%w/v) Beta mercaptoethanol 5.0(%w/v) Components Final concentration (5X buffer) SDS 0.125M Glycine 1.25M Tris HCl 0.5% (w/v) 18
  19. 19. Nelson-Somogyi method  The reducing sugar when heated with alkaline copper tartrate reduce the copper from cupric to cuprous oxide is treated with aersenomolybdic acid, reduction of molybdic acid to molybdenum blue takes place.  The blue colour developed in compared with set of standards in a calorimetric at 500nm  Preparation of different reagents used in NS method.  Dissolve all the components in 100 ml and final volume adjust to 250ml, the solution was filtered (whatman no.1) and stored at 80C. Reagent A Amount Sodium carbonate anhydrous 6.25g Sodium potassium tartrate 6.25g Sodium bicarbonate 5.00g Sodium sulphate anhydrous 50.00g 19
  20. 20. Reagent C Reagent C was prepared in the steps under dark.  Dissolve 2.5g of ammonium molybdate in 45ml dH2O (in 100 ml beaker).  Add 2.1 ml of concentration H2SO4 in another beaker. Dissolve 0.3g of sodium arsenate in 2.5 ml of dH2O.  Mix both solution and make up volume up to 50ml.  Filter it (whatman no.1) under dark condition and store at 37C.  Use this concentration after 24 hour incubation. Reagent D  Mix reagent A and B in 25:1 ratio.  Prepare fresh. Reagent B Dissolve 15g of CuSO4 in 50 ml deionized water. Add one / two drops of concentrate H2SO4. make up volume up to 100 ml filter through whatman no.1 and store at 30C. 20
  21. 21. PROTOCOL Protocol for NS method 10l enzyme(0.014mg/ml) + 50 l of substrate(2%CMC) + 40 l of buffer(20mM citrate sodium buffer, pH 5.8) Add 50 l of substrate (2%CMC) + 50 l of buffer buffer(20mM citrate sodium buffer, pH 5.8) Heating 60°C for 5 minutes Add 100l of NSD (reagent B+ reagent A, 25:1) reagent (vortex and mix properly) Boiling for 20 minutes Add 100l NSC (vortex properly), incubate for 10 minutes Add 700 l dH2O Vortex properly, centrifuge 13,000 rpm for 2 min, take OD at 500nm 100µl sample 100µl blank 21
  22. 22.  Enzyme activity (U/ml) = ∆A500 x C x V = (µ mole/min/ml) 180 x t x v where, ∆A500 = change in absorbance of the sample at 500 nm C = 1 OD equivalent from glucose standard plot (277.77) V = volume of the reaction mixture in ml (0.1ml) t = time of reaction (min) 180 = molecular weight of glucose v = volume of the enzyme taken in assay in ml for reducing sugar estimation(.01ml)  Specific activity = Enzyme activity = U/mg Enzyme concentration  For example at 5 minute in optimized conditions (pH 5.8 and temperature 60°C) the enzyme activity and specific activity with 1%CMC will be Enzyme activity = 0.436×277.77× 0.1 = 12.110 = 1.34 U/ml 180×5×.01 9 Specific activity = 1.34 = 96 U/mg [Enzyme concentration= 0.014 mg/ml] 0 .014 22
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