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In vitro Chaperone Activity Assay Using α-Amylase as Target Protein

Jeeshma Nambidi Parambath Jeeshma Nambidi Parambath
Gayathri Valsala Gayathri Valsala
Karthik Menon Karthik Menon
Shiburaj Sugathan Shiburaj Sugathan

Published: Jun 20, 2018 DOI: 10.21769/BioProtoc.2878 Views: 6725

Edited by: Dennis Nürnberg Reviewed by: Sabu AbdulhameedPooja Saxena

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Abstract

Small heat shock proteins (sHSP) are stress proteins which are ubiquitously found in almost all living organisms. They function as molecular chaperones, which assist in protein folding during translation and in the prevention of irreversible protein aggregation under denaturing conditions. This protocol describes the use of α-amylase as target protein in assessing the chaperone activity of wild and mutant recombinant small heat shock proteins of Mycobacterium leprae. Chaperone activity of these proteins, along with α-crystallin, a standard sHSP was demonstrated using a new method employing their protective effect against heat denaturation of α-amylase from porcine pancreas. The regained enzymatic activity of the α-amylase was demonstrated on starch agar plates stained with Iodine-Potassium Iodide (I2-KI) solution.

Keywords: Small heat shock proteins search sHSP18 search α-amylase search Chaperone assay search Heat inactivation search

Background

Heat shock proteins (HSPs) are a conserved group of proteins which are induced when cells are exposed to external stress including heat and cold stress. Most of the members in this group are functionally related and are involved in the protein folding and unfolding mechanism. Small heat shock proteins (sHSPs) are a subset of HSPs with a molecular size ranging from 12 to 43 kDa and a conserved C-terminal region, called ‘α-crystalline domain’. The sHSPs show ATP independent molecular chaperone activity by binding to partially unfolded proteins and preventing their complete denaturation. There are several methods for demonstrating in vitro chaperone activity of sHSPs, using various substrate proteins like RuBisCO (Goloubinoff et al., 1989), rhodanese (Mendoza et al., 1992), insulin (Farahbakhsh et al., 1995), lysozyme (Rozema and Gellman, 1996), malate dehydrogenase (Lee et al., 1997), citrate synthase (Grallert et al., 1998), xylose reductase (Rawat and Rao, 1998), sorbitol dehydrogenase (Marini et al., 2000) and luciferase (Bepperling et al., 2012) etc. In these assays, the protective activity of sHSPs or other molecular chaperones is demonstrated based on their efficiency in refolding and prevention of aggregation during heat or chemical denaturation. These substrates have different quaternary structures, different rates of folding, and different tendencies to undergo irreversible side reactions during denaturation and HSP assisted renaturation. It has also been shown that protection against heat-inactivated restriction enzymes like NdeI and SmaI can be used to demonstrate the chaperone activity of α-crystallin in vitro (Hess and FitzGerald, 1998; Santhoshkumar and Sharma, 2001). HSP18 is one of the major immunodominant antigens of Mycobacterium leprae, and has been functionally characterized as an sHSP (Lini et al., 2008). In this protocol, we describe a simple method to assay chaperone activity of small heat shock proteins using heat inactivated α-amylase. The efficiency of sHSPs in preventing heat denaturation of α-amylase is demonstrated on starch agar plates after staining with an I2-KI solution. The iodine reagent binds to the starch polymer to form a dark blue color in the starch agar plates. Clear halos surrounding each well with sample are indicative of active amylase enzyme, which digests the starch in the agar plate (Atlas et al., 1995). The advantage of this method is its simplicity and the empirical demonstration of results on starch agar plates.

Materials and Reagents

  1. Conical flasks 250 ml (BOROSIL, catalog number: 4980021 )
  2. Dialysis tubing cellulose membrane (14 kDa cut-off, Sigma-Aldrich, catalog number: D9652 )
  3. Microcentrifuge tubes 1.5 ml, 2 ml, 200 µl (Tarsons, catalog number: 500010 ) 
  4. Parafilm (HiMedia, catalog number: LA017 )
  5. Petri dishes 100 mm, plastic (Tarsons, catalog number: 461030 ), 150 mm, glass (BOROSIL, catalog number: 3160081 )
  6. Pipette tips 1000, 200, 10 µl (Tarsons, catalog numbers: 521020 , 521010 , 520010 )
  7. Polypropylene columns (6 ml) (QIAGEN, catalog number: 34924 )
  8. Test tubes (BOROSIL, catalog number: 9820U04 )
  9. Whatman No.1 filter paper (GE Healthcare, Whatman, catalog number: 1001090 )
  10. 96-well polystyrene flat-bottomed microplate (Tarsons, catalog number: 941196 )
  11. Strains: E. coli strain C41 DE3 (pLysS: F – ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CmR) Lucigen was utilized for recombinant protein expression (Dumon-Seignovert et al., 2004)
  12. Vector: pQE31 (QIAexpress Type IV Kit, QIAGEN, catalog number: 32149 ) 
  13. Molecular chaperones: α-crystallin from bovine eye lens (Sigma-Aldrich, catalog number: C4163 ), recombinant wild and mutant sHSP18 of M. leprae (Lini et al., 2008)
  14. Sterile water (Distilled)
  15. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  16. Isopropyl-β-D-thiogalactoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
  17. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
  18. α-amylase enzyme from porcine pancreas (Sigma-Aldrich, catalog number: A3176 )
  19. Ethanol (70%)
  20. Starch soluble (HiMedia Laboratories, catalog number: GRM3029 )
  21. Bradford reagent (Bio-Rad protein assay dye reagent concentrate, Bio-Rad Laboratories, catalog number: 5000006 )
  22. Tryptone (HiMedia Laboratories, catalog number: RM9111 ) 
  23. Yeast extract (HiMedia Laboratories, catalog number: RM027 )
  24. Agar powder (HiMedia Laboratories, catalog number: GRM026P )
  25. Sodium chloride (NaCl) (HiMedia Laboratories, catalog number: GRM3954 )
  26. di-Sodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) (HiMedia Laboratories, catalog number: RM257 )
  27. Potassium dihydrogen orthophosphate, monobasic (KH2PO4) (HiMedia Laboratories, catalog number: GRM249 )
  28. Sodium dihydrogen phosphate, monohydrate (NaH2PO4·H2O) (HiMedia Laboratories, catalog number: GRM3963 )
  29. Potassium chloride (KCl) (HiMedia Laboratories, catalog number: RM698 )
  30. Imidazole (HiMedia Laboratories, catalog number: RM1864 )
  31. Potassium iodide (KI) (HiMedia Laboratories, catalog number: GRM1086 )
  32. Iodine (HiMedia Laboratories, catalog number: GRM1064 )
  33. TGX FastCast Acrylamide Kit (12%) (Bio-Rad Laboratories, catalog number: 1610175 )
  34. Lysogeny broth (LB) (see Recipes)
  35. Phosphate buffer saline (PBS, pH 7.4) (see Recipes)
  36. Lysis buffer (see Recipes)
  37. Starch agar (see Recipes)
  38. Starch solution (see Recipes)
  39. Iodine-potassium iodide (I2-KI) solution (see Recipes)

Equipment

  1. Analytical balance (Sartorius, model: BSA423S )
  2. Autoclave (Labline, India)
  3. Cooling Centrifuge (HERMLE Labortechnik, model number: Z 323 K )
  4. Deep freezer -80 °C (Labline, model: Ultrafreez ) 
  5. Digital camera (Nikon, model: D5100 )
  6. Incubator (Thermo Scientific Heratherm, model number: IGS60 ) 
  7. Laminar air flow system (Genesys, model: GS LAF 4X2 ) 
  8. Magic Mixer (TOPSCIEN, model: TMM-5L )
  9. Magnetic stirrer (IKA, model: C-MAG HS 7 )
  10. Cork-borer, nickel-plated brass, 6 mm diameter (Sigma-Aldrich, model number: Z165220 ) 
  11. Milli-Q Integral Water Purification System for Ultrapure Water (Merck, catalog number: ZRXQ003WW ) 
  12. Pipettes (1,000 µl, 100 µl, 10 µl and 2.5 µl) (Eppendorf, model: Research® plus )
  13. SDS-PAGE Electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN® 3 Cell ) 
  14. Spectrophotometer (JASCO, model: V-730 )
  15. Water bath (DAIHAN LABTECH, model number: LWB 211D ) 
  16. Microplate reader (Tecan Trading, model: Infinite® M200 PRO )

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Biochemistry > Protein > Activity

Microbiology > Microbial biochemistry > Protein > Interaction

Molecular Biology > Protein > Protein-protein interaction

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