ODODODODODODO □ ODODODODODOD ODODODODODODO DODODODODODOD O D O D O 1-1 ~ ^ O D O D O Kód předmětu: BÍ8980 1 MASARYKOVA UNIVERZITA □ o ÄlNA^^o D ODODOu^uODODO DODODODODODOD ODODODODODODO Protein expression and purification • IX. Protein quality Lubomír Janda, Blanka Pekárová and Radka Dopitová Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. DODODODO ODODODOD D O D O D O D ( MINISTERSTVO ŠKOLSTVÍ, EVROPSKÁ UNIE fc^^^ ■ mládeže a tělovýchovy INVESTICE DO ROZVOJE VZDĚLÁVÁN A* OP Vzdělávání pra konkurenceschopnost m 1 Source of protein-► Extraction-► Separation -► Characterization Quality parameters of produced protein High quality of produced protein 4 3D structure determination Detail function analysis How to determine the protein quality parameters? PROTEIN PURITY = protein is homogenous in its native state How to determine protein purity? > electrophoresis (SDS, native) > gel filtration > mass spectrometry (MS) > dynamic light scattering AHP3 theoretical Mw 22.3 kDa Intact MS analysis PROTEIN PURITY SDS-PAGE > For a pure protein, only one band is expected (5, 10, 20 jig of protein/lane of the gel). > Mw determination (information about protein subunits) > Protein purity is evaluated in SDS-PAGE using various software (e.g. Quantity One from Bio-RAD). > Definition of protein purity: a quantity of particular band as measured by its intensity, expressed as a percentage of the total intensity of all bands in the lane of the gel 28% purity 80% purity 95% purity PROTEIN PURITY > NATIVE PAGE > PFO NATIVE PAGE - The method was developed for analyzing native multimeric structure of many proteins. - PFO (perfluoro-octanoic acid) protects interaction within protein oligomers and enables the determination of molecular mass for multimeric proteins. 15% SDS PAGE AHP3 AHP5 ^> 10% NATIVE PAGE Tween20 NATIVE PAGE x PFO NATIVE PAGE Gel filtration > Gel filtration chromatography separates proteins on the basis of size. > Molecules move through a bed of porous beads. Smaller molecules diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly. > Both molecular weight and three-dimensional shape contribute to the degree of retention. Superdex 75 27 03 2007 Superdex75 1660Valve7001:10 UV Fractions 0Valve7001:10 Inject book 27 03 2007 Superdex75 1660Valve7001:10 P960 Flow Cki rd 23.5 kDa Elution volume 65 ml Elution volume 65 ml suoerdex/5 166U23 U1 U/UU1:1U UV buoerdex/5 166U23 U1 U/UU1:1U Fractions buoerdex/5 166U23 U1 U/UU1:1U Iniecl superdex75 166023 01 07001:10 UV@01,EBASEM miAU 600 d00 400 300 200 100 46.42 DLS is a method to determine hydrodynamic sizes, polydispersities and aggregation effects of protein samples. It can be understood as a method to measure the velocity of the Brownian motion. Intensity at a detector Time -> > The laser is focused in the sample cell and the particles scatter the light in all directions. > Fluctuations in the scattering light intensity are measured as a function of time over a time scale of approximately 100 ns-30 ms. > Those fluctuations are due to the Brownian motion of scattering particles. > The time scale of the scattering fluctuations is directly related to the diffusion coefficient of the scattering particles, which in turn is related to protein size (Stokes-Einstein equation). > In DLS, larger particles produce a very strong scattering intensity signal. Dynamic light scattering (DLS) Dynamic light scattering (DLS) Examples of results How to determine the protein quality parameters? STUDY OF PROTEIN FOLDING INTO THE NATIVE STRUCTURE The atomic resolution structure of a protein can only be revealed by the methods of X-ray crystallography and NMR. - time consuming (not for rapid screening) - not easy to obtain protein in high quality for these types of experiments Methods to study secondary, tertiary, and quaternary structure Circular dichroism Gel filtration ▼ Fluorescence emission spectroscopy Fluorescence emission spectroscopy Amino acids with intrinsic fluorescence properties: phenylalanine, tyrosine, tryptophan; but only tyrosine and tryptophan are used experimentally because their quantum yields (emitted photons/excited photons) are high enough to give a good fluorescence signal. So this technique is limited to proteins having either Trp or Tyr, or both. At an excitation wavelength of 280 nm, both Trp and Tyr will become excited. To selectively excite Trp only, a 295 nm wavelength must be used. Trp and Tyr residues can be used to follow protein folding because their fluorescence properties (quantum yields) are sensitive to their environment, which changes when a protein folds/unfolds. In the native folded state, Trp and Tyr are generally located within the core of the protein, whereas in a partially folded or unfolded state they become exposed to solvent. 1. In a hydrophobic environment (buried within the core of the protein), Tyr and Trp have a high quantum yield and therefore a high fluorescence intensity. In a hydrophilic environment (exposed to solvent), on the other hand, their quantum yields decrease, leading to low fluorescence intensity. 2. The wavelength of the emission maximum also reflects the hydrophobic (l em 308 nm for azurin indicating that Trp is deeply buried within the core of the protein) and hydrophilic (l em 352 nm for glucagon indicating that Trp is exposed to the solvent) environment. Measurement of fluorescence intensity The most common ways of unfolding a protein are using chaotropic agents (urea, quanidin chloride), changing the pH, or increasing temperature. It is possible to measure either steady state or kinetic unfolding. For example, the protein is unfolded by increasing temperature, so at each temperature the protein undergoes unfolding and reaches an equilibrium state corresponding to a partially folded or fully unfolded state depending on the conditions. For each temperature, the fluorescence emission of Trp or Tyr is measured and compare to that native state protein. > Fluorescence intensity (FI) will change upon unfolding. Following the change of this parameter, an unfolding curve is generated by plotting FI = f (temperature). > For kinetic studies, the protein is kept at one temperature and its unfolding reaction is followed in time. Measurement of fluorescence intensity > As mentioned, the previous technique is limited to proteins containing Trp or Tyr residues. It is also possible to use probes that bind specifically to hydrophobic protein residues (Sypro Orange). Those surfaces are hidden in a native protein but exposed in partially folded or fully unfolded proteins. Cf- Ml Tuu CD**} Temperature vs. fluorescence signal format i_—__---1 1429.703 1229.703 1029.703 829.703 G29.703 429.703 229.703 29.703 55 GO Temperature (*C) Circular dichroism spectroscopy - exploring the secondary structure of proteins The technique depends on the difference in absorbance between left and right circularly polarized light beams. CD can be observed in the range of wavelengths at which chiral molecules absorb light. The relevant spectral region is in far UV, i.e. from 240 nm down to 180 nm, where peptide bonds absorb light. In this region, the different types of regular secondary structure, such as alpha-helix and beta-sheet, exhibit a characteristic spectral pattern. Circular dichroism spectroscopy - exploring the secondary structure of proteins 80000 -40000 - I ! I 180 200 220 240 260 Wavelevgth (nm) a-helix spectrum shows the characteristic two negative minima at 208 and 222 nm and a positive maximum at 193 nm. p-sheet spectrum shows a single negative minimum at about 215 nm and a positive maximum at 196 nm (both of these are much smaller than the signal for a-helix). The random coil shows only a very small signal above 210 nm and a small negative minimum at about 198 nm. Circular dichroism spectroscopy - exploring the secondary structure of proteins 1. Maize ß-glucosidase, structure (b/a) 8 barrel Far-UV circular dichroism spectra of wild type b-glucosidase enzyme and its mutants F193A, F200K, W373K, F461L, P2, P3 and P4 3. Far-UV spectra of CGRP (calcitonin gene related peptide) in phosphate buffer of pH 7 (dashed line) and in the same buffer + 50% trifluoroethanol (solid line) 2. AHP proteins, structure 6 a-helix bundle Far-UV circular dichroism spectra of three purification fractions of Arabidopsis thaliana histidine phosphotransfer protein AHP3 eluted at different salt concentrations (B8 - 240 mM NaCl, B9 - 254 mM NaCl, B10 - 260 mM NaCl) 3. 16000 3> -16000 -32000 190 2. 30 25 20 15 10 5 0 -5 -10 -15 -20 180 195 210 225 240 wavelength (nm) 255 210 230 Wavelevgth (nm) 25(" Determination of quaternary structure and activity of p-glucosidase and its mutant forms Gel filtration chromatography may be used to analyze the molecular size of macromolecules. Gel filtration A 25(h 200- I 150 CM I 100 50 WT E<101D F193A F200K W373K F461L Standards 25 50 75 Elution volume (mL) B & i # a ### f ff mdumdrndrndm kDa 66 45 36 3 s Native PAGE Dimer monomer Fig. 5. Quaternary structure of wild-type and mutant Zm-p60.1 p-glucosidases. (A) Elution profiles of wild-type and mutant Zm-p60.1 p-gluco-sidases from the HighLoad 16/60 Superdex 200 column. A sample (1.5 mL) of each enzyme purified by metal chelate affinity chromatography was applied to the column and eluted with elution buffer (50 mrvi Tris/HCl, 500 mivl NaCI; pH 7.00). Fractions corresponding to peaks d and m were collected and analyzed by (B) Coomassie BriilianL Blue-stained SDS/PAGE, (C) Coomassie Brilliant Blue-stained native-PAGE and (D) in-gel activity staining of native-PAGE gels. Peaks 1, 2, 3, 4 and 5 correspond to Blue Dextran 2000; ferritin (Mr440 kDa), aldolase {Mr 158 kDa), BSA (Mr 67 kDa) and ovalbumin (Mr 43 ■cDa), respectively, used as standards. Arrow marks positions of the wild-type and mutant Zm-p60,1 polypeptides in SDS/PAGE. PROTEIN STABILITY B First structurally detailed molecular model of bacterial cytoplasm (combination of proteomic data with high resolution structural data) ■Name Mt» - • Adk :.[ M AhpC 7 • AEd mi 1 * Bcp CspC 7 TU CysK n li m DrjpA 125 : * DiuK 41 TL ttt 20 U Fno 91 1* Fba 7Ü h Frr ■ MisA « Name Miv r t.Jj'A 112 ClnA 611 1 «im M .1 * FA Ii 55 1 * Hm 5 7 15 12 * kdA 43 ilyC H 14 Müll L-5 13 m MelE U 213 m Mop 2 Mw ft PanF HO V i# U :h. • nip 1» Elp PpiR 18 T • PmA 94 4 «• Puii « 7 Fyr 308 * RplA lb Rpo ÍM J * Silt 79 N SodA ■lb n * bJodB li- Figure 1, Thtt^fapljirrt modeL A. Siteiridln uWelVIOiy of TlieO)rrtfiiWS of Tftt iyioplasin MOdeL ELKelVlShiquI TlW .lytoplairn IrtdfriaTttW eiJ d JnyAihian dyftdJrli^ lunnlatiuft infrj Mlh ll-.fi *+idl elWr-jy irtodfil ^S4f4f UMJ. UNA r. IhCWn <±. grdeft altd yfcUd*. I'M* G^ultfWi* plipanfid Wflfc 111 (]]. nail 0.1371 .'y> m i itbi.1 aaaaMjgcn 1 Proteins removed from their natural cellular environment are subjected to a variety of external factors that may lead to a loss (or impairment) of their biological activity as a result of both covalent and non-covalent structural alteration. TJimf Mw m 142 4 Tig ts 9 M TplA H 5 Tfif bl 12 <* TuSA 81 lil Upp ■15 11 UspA 21 7 H)5 [rJÍS ID 305 788 ID 1CKA-C 24 37 IRNA-0 21 37 <* lRrVA-F IS 37 Clf ?.r. 8 PROTEIN STABILITY The shelf life of a protein depends on the intrinsic nature of the protein and on storage conditions. Many factors cause protein inactivation during purification and storage: > Nearly every property of a protein, except for its primary amino acid sequence, can be shown to vary with the solution conditions, such as pH, temperature and ion concentration. > Loss of an essential cofactor. > Exposure to proteases, oxygen, or heavy metals. > Changes in physical conditions such as freezing and thawing. CHEMICAL REACTIONS AFFECT PROTEIN STABILITY Succinimide (cyclic imide) formation following deamination Formation of p-isoaspartate as a result of deamination of asparagyl residues or isomerization of aspartyl residues is a major source of instability in proteins and peptides, especially at neutral and alkaline pH after prolonged storage. -HE H, Ů B Bu ccim nuda UftürtfH 1 : X H -OH [Asp) -H {QM -NH, (AstiJ -CK, JAIbJ -OCH3 (Aap (3-mslfiyl ttLäfl -CH,OH |5ar) 3 For instance, the disposition of an asparagine residue followed by a small hydrophilic amino acid residue such as glycine, serine or threonine in a polypeptide chain can lead to deamination of an asparagine and formation of a-aspartic acid and/or p-isoaspartate via an intramolecular rearrangement that produce a succinimide (cyclic imide, unstable under physiological conditions) intermediate. This phenomenon occurs at a rapid rate and conveys extra negative charge to the protein. p-isoaspartate-bearing protein are reported to be specific substrates for a widely distributed methyltransferase enzyme that uses S-adenosyl-L methionine as its methyl donor. It has been proposed that this class of methyltransferases specifically recognizes proteins with incurred chemical changes (p-isoaspartate formation) and that the methylation reaction may be the first step in protein degradation. CHEMICAL REACTIONS AFFECT PROTEIN STABILITY Aspartyl-prolyl bond cleavage Specific cleavage at Asp-Pro peptide bonds in a polypeptide chain can occur by exposure to acid conditions (e.g. 10% acetic acid with pH 2.5, 70-75% formic acid) at moderate temperatures (37°C, 40°C) for periods u p to 120 hours. Pyroglutamate formation Cyclization of amino terminal glutamine residues to pyroglutamyl residues under mild acidic conditions is a frequent cause of protein modification. CHEMICAL REACTIONS AFFECT PROTEIN STABILITY Oxidation Oxidation of proteins by molecular oxygen during isolation and storage can lead to their inactivation and/or aggregation. Cysteine is particularly prone to oxidation, as are methionine and tryptophan residues. Reducing agents that are primarily used to protect free SH-groups from oxidation (particularly of cysteine and methionine residues): dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), £r/s(2-carboxyethyl)phosphine (TCEP). ^ Oxidated protein R-5S-R * HE-CHj(CHaMJL,CH;-SH =S; AS* ■+ P-SS-CKitCH|>lkCHa*5H pj -► 2-ME S-R HOH -► DTT pn^jCOO^jjP: 1 qE3R 1 tV3 —*^ tCHjCH:C[X]|-0jP=O 4 2RSH J5j -► TCEP CHEMICAL REACTIONS AFFECT PROTEIN STABILITY Oxidation It is advisable to add the metal chelating agent EDTA (or EGTA) to protein solution, as well as a reducing sulfhydryl reagent, for complex trace metals such as Cu, Fe, and Zn. These trace metals can either bind to sulfhydryl (cysteine) residues directly or catalyze their oxidation by molecular oxygen. > DTT is preferred to 2-mercaptoethanol, because DTT reduces disulfides quantitatively (does not form mixed disulfides). > The higher the pH and temperature, the shorter the half-lives of the thiol reagents. TABlt A3.4, Hal(-iife "f thiol compound* in solution Conditions pH fi.5,M*C pH 7.5, UPC pHÍ.S. WC rH S.5. trc pH Íi. WZ, pit U.S. J0*C t 0.1 mHOi1' f)H S.i, WC ť !,□ mM EDTA Half-life (hour*) 1 -MeitipHKlh anal DT7 Glutathione '.! -Menrí piopropiúnate ■So IS 7 IŮ 10 i 1-4 i :■ 21 II ří 1 !■ ÍL1 PÍ 0.6 12 «4 4 70 >10Ů Biolojsyl). All Uiiul wnipminds w*rc (lis«J™J in fl.l m potiisium |)^l^MplliM, huffin DTT is stable 1 week at 2-8C, pH 7,75, protected from atm ospheric oxygen. > TCEP is a more stable, faster and stronger reductant than DTT at pH values below 8.0. It resists air oxidation and is stable over a wide pH range (1.5-8.5). > TCEP is recommended for long-term storage of proteins in the absence of metal chelators (EDTA, EGTA), because Fe3+ and Ni2+ catalyzes DTT oxidation. PROTEOLYSIS DURING PURIFICATION AND STORAGE A simple test for proteolytic degradation is to incubate the sample extract or partially purified target protein at 30-37°C, withd rawing aliquats at various time points and assaying them for biological activity. Caution must be exercised with this method of proteolysis detection, as the target protein may be partially degradated, yet retain full biological activity. Therefore, protein size and microheterogeneity have to be monitored by SDS-PAGE and following western blotting and/or by mass spectrometry. If loss of activity occurs upon prolonged incubation at moderate temperature (e.g. 30°C), yet the size of the protein remains unaltered, the structural integrity (e.g. folding) should be investigated using fluorescence, circular dichroism, etc. PROTEOLYSIS DURING PURIFICATION AND STORAGE If loss of biological activity indicates that proteolysis is a problem, then protease inhibitors must be added to the sample and during all future attempts at purification of that target protein. Table 2. Common Protease Inhibitors Protease Inhibitor Target Protease Working Concentration PMSF (Phenylmethylsulfonyl fluoride) Serine proteases 0.1-1 mM Benzamidine Serine proteases 1 mM Pepstatin A Acid proteases 1 |ig/ml Leupeptin Thiol proteases 1 |ig/ml Aprotinin Serine proteases 5 |L i a/ml Aritipain Thiol proteases 1 |Ug/ml EDTA and EGTA Metal lop rote a ses 0.1-imM PROTEOLYSIS DURING PURIFICATION AND STORAGE Protein was co-purified with traces of metalloproteases. PROTEIN CONCENTRATION AFFECTS STABILITY > In general, proteins are less stable at low concentrations (<50 jog/ml). Under these conditions, multiple subunit proteins and cofactors tend to dissociate and physical losses due to adsorption to surfaces can become significant. > When total protein concentration falls below ~50 jog/ml, protein loss can occur due to strong and irreversible adsorption of the protein to a variety of surfaces, including glass, plastic, and various types of filtration media used for concentrating, clarifying, or sterilizing proteins. > It is advisable to keep protein concentration as high as possible (e.g. >1 mg/ml) during purification and storage. This is relatively easy to achieve during the early stages of a purification procedure; however, it becomes more difficult during the later stages of purification or in the case of purification of low abundant proteins isolated from natural sources. In these circumstances, it is imperative that solvent additives, such as polyethylene glycol (0.05% v/v) and nonionic detergents (0.02% w/v, Triton-X-100, or Tween-20), be included in all buffers and eluents used in the purification procedure. > Protein solutions lower than 1 mg/ml should be concentrated as rapidly as possible using methods such as dialysis and lyophilization. > In situations where rapid concentration of a dilute protein solution is not possible and the presence of an exogenous protein can be tolerated, the addition of ~1 mg/ml bovine serum albumin (BSA) has been shown to be very effective. > On the other hand, protein aggregation is generally concentration dependent. Protein aggregation - a major event of physical instability of proteins > Under certain conditions (or simply with time), the secondary, tertiary and quaternary structure of a protein may change and lead to protein unfolding and/or aggregation (the assembly of individual protein molecules into amorphous, multimeric states). > Protein aggregates may have reduced or no activity, solubility, and altered immunogenicity. Mechanism of protein aggregation ■ Proteins aggregate to minimize thermodynamically unfavorable interactions between a solvent and exposed hydrophobic protein residues. ■ Hydrophobic interaction, i.e. the reluctance of non-polar groups to be exposed to water, is considered to be a major driving force for both protein folding and aggregation. Both protein aggregation and folding represent a balance of exposed and buried hydrophobic surface areas. The balance is so delicate that a change of one amino acid in a protein may substantially change its aggregation behavior. ■ Other possible mechanisms: formation of non-native disulfide bonds, electrostatic interactions..... ■ Protein aggregation may be induced by a variety of physical factors, such as temperature, ionic strength, foaming, protein concentration, pH shift, vortexing, etc. These factors can increase the hydrophobic surface of proteins, causing aggregation. STABILIZATION IS NECESSARY FOR LONG-TERM STORAGE OF PROTEINS Solvent additives that stabilize the protein: Osmolyte stabilizers = a class of weakly charged compounds with low molecular weight that are used as co-solvents for stabilizing purified proteins for storage and during freezing, thawing and lyophilization. They enhance stability through a preferential hydration mechanism as well as a solvophobic effect (TMAO). > Osmolyte stabilizers include sugars (glycerol, xylitol, PEG), amino compounds and small neutral amino acids (glycine, alanine), including their derivatives, as well as large dipolar molecules such as trimethylamine N-oxide (TMAO). STABILIZATION IS NECESSARY FOR LONG-TERM STORAGE OF PROTEINS Solvent additives that stabilize the protein: Ionic stabilizers > Certain ionic compounds and neutral salts can stabilize proteins in solution. > The stabilizing effect of neutral salts varies with the position of the constituent ions in the Hofmeister lyotropic series, which reflects ionic effects on protein solubility, association-dissociation equilibria, and enzyme activity. The Hofmeister series ranks both cations and anions according to their stabilizing effects. HOFMEISTER SfcRlES In the HaJmHSteF series, the most sdibrtizin^ fonu (saltinjj-oui ions} are on the left jnd the mnsi cJesta-biJizirtg iofii (salting tons) are on (he nigjit: 5+ In general, stabilizing ions favor the ordered or folded (native) conformation of a protein in aqueous solution by strengthening intramolecular contacts. This helps to prevent the unfolding process, the initial event in protein aggregation/inactivation. Usually, salts that destabilize the form of a protein increase its solubility in water of the hydrophobic amino acid side chains that normally exist in the "interior" of an ordered (native) protein structure. i-: si .iliIlijvr (itrSle, tfulfoneSf acetate, plwvphn.ie.t qu^erturf aniLn« IlITHl t linJu di(M< in?(r*ftion wiLh bill aJBfCC ihf tnilli inLu.1ii.in jwuptftlei In HTjitr. The** a.KiM.1 unlYLIK HaaUKIA J IWjrstAqliKin|(.4ilJ&.LS Oil prwclas WSSUt within a LuiiLeiLl.rai.ii.il i uugr n'Wrli eiviiCipmj lIlii-i (hjh Initial use of a weak ionic stabilizer, such as guanidinium acetate (to arrest protein unfolding), followed by slowly changing to an intermediate stabilizer (e.g. guanidinium sulfate) and finally to a strong ionic stabilizer (e.g. sodium sulfate) allows for a protein with intermediate folded forms to return gradually to a more native-like structure. STABILIZATION IS NECESSARY FOR LONG-TERM STORAGE OF PROTEINS Low temperature storage > Although chemical modification and/or proteolytic degradation of proteins can occur at moderate temperatures, the extent of these changes is impaired at increased temperatures. > High number of commercially available biochemicals that are supplied in >3M ammonium sulfate or ~50% glycerol. Typically, proteins stabilized in this manner should be stored at refrigerator temperatures (4-6°C), although more labile proteins should be stored at -20°C and extremely labile proteins at even lower temperatures (- 70°C). >It is important to determine the proper storage conditions for the target protein because certain proteins (so-called "cold labile" proteins) are more stable at ambient temperature than in a refrigerator. Cold denaturation of proteins (i.e. unfolding of proteins at low temperatures) has been well characterized and has been shown to be an inherent property of the protein itself (and distinct from "freezing inactivation"). STABILIZATION IS NECESSARY FOR LONG-TERM STORAGE OF PROTEINS Comparison of protein storage conditions Storage conditions Characteristics Solution at 4°C Solution in 2550% glycerol or ethylene glycol at -20°C Frozen at -20C to -80°C or in liquid nitrogen Lyophilized (usually also frozen) Typical shelf life 1 month 1 year Years Years Requires sterile conditions or addition of antibacterial agent Yes Usually No No Number of times a sample may be removed for use Many Many Once; repeated freeze-thaw cycles generally degrade proteins. Once; it is impractical to lyophilize a sample multiple times. STABILIZATION IS NECESSARY FOR LONG-TERM STORAGE OF PROTEINS General considerations for protein storage > Store the protein in a concentration of at least 1 mg/ml. > If the concentration is lower, stabilize the protein by adding another protein, e.g. BSA, or certain additives. Many compounds may be added to protein solutions to lengthen shelf life: > Cryoprotectants such as glycerol or ethylene glycol added to a final concentration of 25-50% help to stabilize proteins by preventing the formation of ice crystals at -20°C that destroy protein structure. > Protease inhibitors prevent proteolytic cleavage of proteins. > Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.020.05% (w/v) inhibit microbial growth. > Metal chelators such as EDTA at a final concentration of 1-5 mM prevent metal-induced oxidation of SH groups and help to maintain the protein in reduced state. > Reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at a final concentration of 1-5 mM also help to maintain the protein in the reduced state by preventing oxidation of cysteines.