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C H A P T E R E I G H T E E NAdvances in Preparation of Biological Extracts for Protein PurificationAnthony C.GrabskiContents1.Introduction2862.Chemical and Enzymatic Cell Disruption2863.Mechanical Cell Disruption2904.Concluding Remarks2935.Procedures,Reagents,and Tips for Cell Disruption2935.1.Buffer composition2935.2.Cell disruption buffers2965.3.Microscale protocols for E.coli cell lysis2965.4.Gram-scale mechanical disruption of E.coli299References301 AbstractThere are a variety of reliable methods for cellular disintegration and extraction of proteins ranging from enzymatic digestion and osmotic shock to ultrasonica-tion,and pressure disruption.Each method has inherent advantages and disadvantages.Generally vigorous mechanical treatments reduce extract vis-cosity but can result in the inactivation of labile proteins by heat or oxidation, while gentle treatments may not release the target protein from the cells,and resulting extracts are extremely viscous.Depending on the cell type selected as the source for target protein expression,cellular extracts contain large amounts of nucleic acid,ribosomal material,lipids,dispersed cell wall polysaccharide, carbohydrates,chitin,small molecules,and thousands of unwanted proteins.Isolation and recovery of a single protein from this complex mixture of macro-molecules presents considerable challenges.The first and possibly most impor-tant of these challenges is generation of a cellular extract that can be efficiently manipulated in downstream processes without inactivation or degradation of labile protein targets.Cell disruption techniques must rapidly and efficiently lyse cells to extract proteins with minimal proteolysis or oxidation while reduc-ing extract viscosity caused by cell debris and genomic DNA contamination.Department of Research and Development,Semba Biosciences,Inc.,Madison,Wisconsin,USAMethods in Enzymology,Volume463#2009Elsevier Inc. ISSN0076-6879,DOI:10.1016/S0076-6879(09)63018-4All rights reserved.285286Anthony C.Grabski Advanced bioprocessing equipment and reagents have been developed over the past twenty years to complement established disruption procedures and accomplish these tasks with even greater success.This chapter will summarize these advances and describe detailed protocols for some of the most popular methods for protein extraction.1.IntroductionEarly protein chemists worked primarily with small extracellular proteins that were stable,plentiful,and easily isolated without cell disrup-tion.Intracellular proteins expressed at physiological levels might constitute <0.001%of the total cellular protein and were difficult to extract and recover without proteolysis or loss of enzymatic activity.Modern recombinant protein production through genetic engineering of Escherichia coli can result in expression of the target protein at over40%of the cellular total.Recombinant yeasts such as Saccharomyces cerevisiae,Pichia pastoris,and Kluvyeromyces lactis are capable of expressing complex proteins requiring posttranslational modifications(Spencer and Spencer,1997).Recovery of the intracellular recombinant proteins from these hosts requires effective cell disruption and extraction techniques.Well established protocols have been reviewed and detailed procedures described for laboratory and process scale disruption of various cell types by both mechanical and nonmechanical methods(Andrews and Asenjo,1987;Cull and McHenry,1990;Dignam, 1990;Engler,1985;Gegenheimer,1990;Harrison,1991;Hopkins,1991; Jazwinski,1990;Middelberg,1995).Radical changes to standard disruption equipment and procedures had not occurred up to1995(Foster,1995). However,the evolution of structural and functional proteomics demanded new reagents and automated methods to streamline the process steps converting gene sequences to purified proteins(Grabski et al.,2002).Proteomics and structural genomics efforts require cell extraction meth-ods that allow screening of multiple host vector combinations for hundreds of proteins in parallel(Stevens et al.,2001).The best combinations for correctly folded high-level expression identified in these multiparallel screens are scaled up in order to produce milligram quantities of purified protein for functional and structural analysis(Dieckman et al.,2002;Lesley, 2001;Stevens and Wilson,2001;Zhu et al.,2001).These high-throughput (HT)protein expression and purification strategies have fostered develop-ment and optimization of reagents and instrumentation that allow efficient cell lysis and protein extraction at both micro-and macroscales.Preparation of Biological Extracts2872.Chemical and Enzymatic Cell DisruptionMicro-scale cell disruption is often accomplished through chemical and enzymatic methods or combinations of the two.Lysis methods such as sonication and French press are not easily applied to cell pellets harvested from5ml of culture or less,and excess heat and oxidation are common problems.A significant advancement for simplification of microscale cell lysis and has been the development of detergent-based reagents such as B-PerÒ(Chu et al.,1998)and BugBusterÒ(Grabski et al.,1999).These reagents do not require expensive equipment,are very fast and easy to use,and are the most practical and effective means to HT cell extract preparation.Highly active enzymes and enzyme mixtures have also been developed to improve lysis efficiencies and decrease extract viscosity by completely digesting geno-mic ed alone and in combinations these commercially available lysis reagents and enzymes(Table18.1)efficiently disrupt cells and extract pro-teins from bacteria,yeasts,plants,insect cells,and higher eukaryotes.Deter-gent-based methods for extraction and enrichment of proteins and subcelluar organelles from eukaryotic cells and tissues are covered extensively in chapter 19by Michelsen and von Hagen of this volume.Another advancement in chemical cell disruption tailored for HT robotic processing of samples,is the development of reagents for extraction of recombinant proteins from E.coli without cell harvest from the culture media(Grabski et al.,2001,2003;Stevens and Kobs,2004).Traditional protein purification methods require an initial cell harvest step,which concentrates cell mass and removes spent media components(Burgess, 1987;Deutscher,1990;Scopes,1994).This step,as well as any mechanical lysis step is difficult to automate and scale down for parallel extraction and purification of small amounts of proteins simultaneously.Concentrated, detergent-based reagents(Table18.1)such as PopCulture TM,FastBreak TM, and B-PERÒDirect,combined with high-activity lysozyme and nuclease overcome these bioprocessing hurdles to automation.The advantages of these reagents for HT extraction and purification are eliminating the need for multiple centrifugation steps to separate cells from culture media and clarify the crude extract,and eliminating the need for mechanical disruption (Nguyen et al.,2004).These innovations allow direct affinity adsorption of target proteins from the total culture extract,and the entire cell growth, extraction,and purification can be done in a single tube or well.The active ingredients in many of the bacterial lysis reagents are nonionic or zwitterionic detergents functioning to disrupt cell membrane and cell wall structures weakening the cells for rupture by osmotic shock,Table18.1Commercial cell disruption reagents and enzymesVendor Reagents Cell types Vendor web siteEMD Chemicals-Novagen BugBusterÒ,YeastBuster TM,Bacteria,yeast,insect,mammalianwww.emdbiosciences.comCytoBuster TMPopCultureÒrLysozyme TM,BenzonaseÒ,Lysonase TMM-Pek,S-PekEpicentre EasyLyse TM,OmniCleave TM,ReadyLyse TM Bacteria Promega FastBreak TM Bacteria Roche Complete TM Lysis(B,M,Y)Bacteria,mammalian,yeast www.roche-applied-Semba Biosciences, Inc.Recombinant Lysozyme,BenzonaseÒ,Liquisonic TMBacteria Sigma Aldrich CelLytic TM(B,IB,M,MEM,MT,NuClear TM,P,PN,and Y)Bacteria,mammalian,plant,yeastwww.sigmaaldrich.comThermo Fisher-Pierce POP-PERSÒ(B-,I-,M-,NE-,P-,andY-PerÒ)Bacteria,insect,mammalian,plant,yeastwww.thermofisher.comPreparation of Biological Extracts289 freeze-thaw,or enzymatic attack by phage or chicken egg white lysozyme. Gram positive bacteria are easily disrupted by lysozyme treatment alone, with a few exceptions(Cull and McHenry,1990).Gram negative bacteria are difficult-to-disrupt with lysozyme alone because their outer lipid bilayer must be permeabilized first to expose the peptidoglycan cell wall to lyso-zyme digestion.Tris buffer plus EDTA liberates about50%of the poly-anionic lipopolysaccharide in the bilayer(Lieve,1974),but EDTA interferes with the most popular downstream purification step for recombi-nant proteins,immobilized metal affinity chromatography(IMAC).The detergent lysis reagents are extremely effective for outer membrane per-meabilization without IMAC interference.However,detergent-based reagent cell disruption has several disadvantages.Solubilities for some pro-teins extracted with detergents may be enhanced by detergent–protein interactions,and protein solubilities will change depending on the detergent–protein ratio.The solubility of a particular protein extracted by these reagents may not correlate with the protein’s solubility as extracted by scale-up mechanical methods.Detergents and detergent impurities can inter-fere with downstream processing and structural characterization(Swiderek et al.,1997),and fractionation methods that depend on hydrophobic interac-tion should be avoided(Marshak et al.,1996).Despite these drawbacks, detergent-based lysis reagents are widely used in modern proteomics.The exact concentrations and types of detergents employed in these lysis reagents are proprietary and the cellular architecture to be disrupted dictates the chemical properties and concentration of detergent required for the task. Several references from this chapter(Neugebauer,1990;Chu and Mallia, 2001;Eshaghi et al.,2005;Kashino,2003)on applications of detergents for protein extraction and solubilization provide a wealth of information allowing home brewed extraction reagents to be formulated with reasonable success.Yeasts are more difficult-to-disrupt than bacteria.Their thick complex cell wall can compose up to25%of the cells’dry weight.Typical compo-nents are glucans,cellulose,mannoproteins,and chitin interconnected by covalent,disulfide,hydrogen,and hydrophobic bonds.Yeast cells should be harvested in late log or early stationary phase,for more efficient disruption. Yeasts cultured longer into stationary phase develop thicker cell walls and multiple budding scars or aggregates in the case of budding yeast(Catley, 1988).Protease inhibitors should be included in lysis buffers or added to commercial lysis reagents and protease deficient strains used for expression. Yeast lysis with reagents such as Y-PerÒand YeastBuster TM(Table18.1) and lysis by controlled manipulation of genes involved in cell wall biogene-sis have provided new tools to complement previously developed mechan-ical and enzymatic methods(Drott et al.,2002).Enzymatic treatment of microbial cells for lysis and viscosity reduction has several distinct advantages over mechanical and chemical methods. The lytic enzymes are highly specific for targeted cell wall components,290Anthony C.Grabski they are gentle and do not generate shear,high temperature or oxidative damage,and they are simple to use requiring no specialized equipment for processing.Enzyme treatments of cells and extracts are also combined with mechanical disruption methods to increase the selectivity of product release, to increase the rate and yield of extraction,to minimize product damage, and to reduce viscosity for downstream processes(Andrews and Asenjo, 1987;Grabski et al.,1999).Improved bioprocessing enzymes and enzyme mixtures(Table18.1)include high-specific activity phage lysozymes such as rLysozyme TM,Ready-Lyse TM,and Recombinant Lysozyme,nonspecific nucleases like BenzonaseÒand OmniCleave TM,and the lysozyme-nuclease cocktails Liquisonic TM,Lysonase TM,and EasyLyse TM.Recombinant Lyso-zyme,Ready-Lyse TM and rLysozyme TM phage lysozymes have specific activities over200-fold greater than that of chicken egg white lysozyme. The nuclease of Serratia marcesens,available as a highly purified recombinant enzyme BenzonaseÒ,is one of the most promiscuous nucleases known.The enzyme cleaves all forms of DNA and RNA(Meiss et al.,1995;Nestle and Roberts,1969).Compared to bovine pancreatic DNase I,Benzonase attacks substrates more evenly making it an excellent choice for reducing extract viscosity caused by nucleic acids.ZymolaseÒand Lyticase glucanases are enzymes useful for yeast protoplasting or yeast cell lysis.Potential problems with enzymatic treatment limit its use especially for industrial cell disruption.These problems include the added lytic enzyme and impurities in the enzyme preparation that complicate downstream purification,degradation of the recovered product during lysis,tempera-ture,and pH optima for the lytic enzyme may be incompatible with the target protein,and the expense and limited availability of most lytic enzymes prohibits their use at an industrial scale(Hopkins,1991).3.Mechanical Cell DisruptionSonication and high-pressure disruption have been effectively applied for disruption of microorganisms,plants,and animal cells.These methods have been used for decades and the equipment and mechanisms involved described in detail(Harrison,1991;Hopkins,1991;Middelberg,1995). Sonication is based on the shear forces created by high frequency ultrasonic vibrations generated by resonation(15–25kHz)of a tuned probe or horn.The sonic pressure waves created cause the collapse of formed microbubbles and their implosion generates shock waves with sufficient energy to disrupt cell walls,and reduce viscosity by shearing nucleic acids.High-pressure homogenizers and pressure extruders operate by forcing a pressurized cell suspension through a narrow orifice valve.The valve may be a simple restricting needle valve as in the French Press or a more complexPreparation of Biological Extracts291 design with a combined valve seat and impact ring found in the APV Manton-Gaulin homogenizer.The mechanism for disruption is a combined pressure drop and shear at the nozzle in pressure extrusion.Numerous mechanisms for cell disruption with pressure homogenizers have been proposed including turbulence,cavitation,viscous shear,and impingement (Kleinig and Middelberg,1998;Pandolfe,1999).Universal acceptance for a single mechanism has not been reached.However,cavitation and impinge-ment have been reported as the major forces responsible for disruption (Shirgaonkar et al.,1998;SPX Corp.,2009).Cell disruption using glass beads to grind the cells in suspension,also known as bead milling or bead homogenization,is a frequently used procedure at both laboratory and production scale.Bead milling can be accomplished with laboratory equipment as simple as a magnetic stirrer, vortex mixer,or blender and with commercial equipment specialized for the process in the form of high speed mills,agitators,and mixers.The cell disintegration is dependent on cell concentration,bead size and composi-tion,ratio of beads to suspension,processing duration,and the forces applied(Ramanan et al.,2008).The method is effective with difficult-to-disrupt cells like yeasts,spores,and microalgae.It is used for bacteria, plant and animal cells and is a preferred method for large-scale disruption of fungi(Hopkins,1991).The mechanism and models of the bead milling process have been reviewed(Harrison,1991;Middelberg,1995),but basi-cally the cells are crushed,ground,and torn apart by abrasive contact and shear forces created between the beads,cells,and reaction chamber itself.The specialized equipment necessary for mechanical cell disruption techniques has undergone functional innovation and new instruments have been developed.The majority of mechanical methods are not easily applied to cell pellets harvested from5ml of culture or less,and excess heat and oxidation are common problems with mechanical disruption.How-ever,96-well sonicator heads and microplate horns are available(Misonix, Inc.Farmingdale,NY;)and for cases where traces of detergent in the purified protein preparation might interfere with biochem-ical characterization or crystallography,this HT physical method is a viable option.The SonicMan high-throughput sonication system(MatriCal,Inc. Spokane,WA;)is a stand alone or integrated unit for 96-,384-,and1536-well plate sonications.This touch screen controlled microplate sonicator uses disposable gasketed pin lids to prevent well-to-well cross-contamination and has a plate shuttle allowing direct integra-tion into robotics platforms.Another new sonicator available from BioSpec Products is the cordless handheld Sonozap Ultrasonic Homogenizer.The 1/8in.diameter auto-tuned probe is ideal for small samples of0.3–5.0ml. Pressure Biosciences,Inc.()has developed the Barocycler TM,bench top,and PCT Shredder,hand held,sample preparation systems.These instruments are capable of rapid,high-pressure292Anthony C.Grabski (up to35kpsi)cycling using specialized PULSE TM tubes.The1.2–1.5ml tubes contain a ram and pored lysis disk that facilitate the hydrostatic pressure induced lysis of plant,animal,insect,or microbial cells(Garrett et al.,2002).The FastPrep(MP Biomedicals,Irvine,CA;www.mpbio. com),Geno/Grinder(SPEX Certiprep,Inc.,Metuchen,NJ;www. )MagNA Lyser(Roche Diagnostics,Penzberg,Germany; ),Mikro-Dismembrator(Sartorius Stedim Biotech, Aubagne,France;),Mini-Bead Beater(BioSpec Products,Bartlesville,OK;),and Retsch Mixer Mill(Retsch GmbH,Haan,Germany;)are all shaking-type bead mills that process multiple milliliter size samples.These devices are not truly HT but are capable of high-yield disruption results from recalcitrant cells in1–5min.The Microfluidics Microfluidizer(Newton,MA;www. )differs from other high-pressure homogenizers. The instrument pressurizes and accelerates split streams of the cell suspen-sion by gas driven pumping of the liquid through fixed-geometry micro-channels.The two high-velocity streams directly impinge on each other creating very high shear forces and pressure drop as the suspension exits the device.Microfluidizer models range from the M-110P capable of processing small sample volumes of25ml and ideal for laboratory use to the M-700biopharmaceutical process models with up to900l/h through-put,complete process control monitoring,CIP,and validatable under21 CFR to cGMP.Constant Systems Ltd.(Low March Daventry,Northants, England;)has designed hydraulically operated disruption instruments that function similar to the original French press by exerting the disruptive forces of pressure extrusion but under controlled, contained and repeatable conditions.The Constant Systems instruments are available in single shot bench(1–20ml)to continuous process scale (405–565ml/min.)eful features include touch screen moni-toring and control,cooling jacket,and CIP/SIP processes.Avestin, Inc.()offers high-pressure EmulsiFlex homogenizers capable of cell disruption from1.0–1000l/h.The homogenizers generate disrupting pressure from500to30,000psi using either gas or electrically driven piston pumps.They can be equipped with heat exchangers for cooling cell extracts and are SIP sterilizable for GMP manufacturing.The BioNeb cell disruption systems of Glas-Col,LLC(Terre Haute,IN; )employ nebulizing gas pressures from10to250psi to break open cells without heat minar flow in the nebuli-zation channel creates cell disrupting shear forces.The magnitude of the force depends on the pressure applied,type of gas(argon<nitrogen <helium)and viscosity of the liquid.The smaller the nebulized droplets, the higher the viscosity,and the greater the gas pressure applied,the greater the shear force created(Surzycki et al.,1996).Preparation of Biological Extracts2934.Concluding RemarksThe cell disruption methods,reagents,and instrumentation described in this chapter are useful and effective if appropriately modified for different cell types and scales,but the question of which method is superior for each application has certainly been asked.Studies have been conducted compar-ing methods of microbial disruption and protein quantitation in resulting extracts(Benov and Al-Ibraheem,2002;De Mey et al.,2008;Guerlava et al.,1998;Ho et al.,2008).The mechanical methods of high-pressure homogenization and bead milling are favorite methods at large scale because of their effectiveness and ability to rapidly handle various process volumes at low cost.Sonication,chemical reagents including detergents,enzymatic treatments,freeze-thaw,and enzymatic plus chemical or physical method combinations are also very effective and are used more frequently at labora-tory and especially microscale.The uniqueness of proteins and differences in host cell structure force a high degree of empiricism in selection and optimization of cell disruption and extract preparation techniques. However,the tools and methods for success in biological extract prepara-tion are available and have kept pace with the demands of modern structural and functional proteomics.5.Procedures,Reagents,and Tips for CellDisruptionThe following strategies and guidelines serve as starting points to produce quality cellular extracts suitable for most downstream purification and analysis processes.Although the focus is on disrupting E.coli at micro-and macrolaboratory scale,many of the techniques can be applied to disrupting and isolating intracellular proteins from other sources and at larger prehensive references for protein purification provide additional information on some of the guidelines for protein extract prepa-ration outlined here as well as on many important aspects of protein purification and characterization(Burgess,1987;Deutscher,1990;Harris and Angal,1989;Hopkins,1991;Marshak et al.,1996;Scopes,1994).5.1.Buffer compositionThe components and volume of cell disruption buffers are critical not only for efficient disruption but will also affect subsequent purification steps and the target protein’s stability and recovery after it has been released from the cells.Each protein extracted is an individual and ideally would have an294Anthony C.Grabski extraction and purification buffer tailored specifically for its own biochemi-cal requirements and intended direction through the purification pipeline. In most cases a more generic extraction buffer can be used with good results if a few basic criteria are met.These criteria are pH,ionic strength,additives to prevent degradation and improve stability,and buffer to cell paste ratio.A very good reference for maintenance of active enzymes through buffer composition and other means is Scopes(1994;‘‘Protein Purification’’). Technical information on pH and buffers including tables for preparation of pH1–13buffers,buffer properties,and the influence of salt,temperature, and dilution values can be found by Dawson et al.(1986).The pH selected for the extraction buffer should be at least one pH unit above or below the protein’s isoelectric point.This pH difference from the pI will prevent isoelectric precipitation by maintaining a positive or nega-tive charge on the protein and also facilitate ion-exchange purification as a purification step.The buffer ionic strength should be20–50m M with a p K a within0.5unit of the desired pH in order to maintain buffering capacity with minimal conductivity increase.The ionic strength inside the cytoplasm of a typical cell is150–200m M with very high concentrations of charged biomolecules for ionic protein interaction.The lysis buffer should contain at least50–100m M NaCl.Increased ionic strength of the extraction buffer will reduce these ionic interactions and precipitating losses from charged particulates that would adsorb protein and be removed by centrifugation or filtration steps.Finally,buffer components to prevent degradation and improve stability should be added as necessary.These include protease inhibitors(Table18.2),reducing agents such as dithiothreitol(DTT),tris (2-carboxyethyl)phosphine(TCEP),or tris(hydroxypropyl)phosphine (THP),divalent cations,cofactors,and kosmotropes like glycerol,sorbitol, or trehalose.The water soluble odorless phosphines TCEP and THP are more stable and effective for the maintenance of reduced protein disulfide bonds than the more commonly used thiol reductants2-mercaptoethanol and DTT(Cline et al.,2004;Getz et al.,1999;Han and Han,1994). Nonionic or zwitterionic detergents may be added to increase solubility of hydrophobic proteins.Reducing agents,protease inhibitors,and deter-gents may interfere with some purification and detection methods and assays.The potential interference of these buffer components must be considered when selecting the chemicals and concentrations employed.A very versatile buffer(Buffer B)for bacterial cell disruption is50m M Tris/HCl or sodium phosphate pH7.5–8.0,50m M NaCl,5%glycerol, 0.5m M EDTA,and0.5m M DTT.Buffer formulations recommended for yeast disruption(Buffer Y)and insect cell disruption(Buffer I)are given below.The volume of buffer used for resuspension of cell pellets must be at least three times the volume of the original pellet for effective disruption and good recovery of the liquid fraction after removal of insoluble cellTable18.2Protease inhibitorsInhibitor Proteases inhibited Stock solution Effective concentration Aprotinin Serine10mg/ml PBS0.6–2.0m g/ml Benzamidine Serine50mg/ml water0.5–4.0m ME-64(L-transepoxysuccinyl-leucylamido-Cysteine 5.0mg/ml water 1.0–10m M [4-guanidino]butane)EDTAMetallo 1.9g/10ml water 1.0–10m M (ethylenediaminetetraacetic acid)Leupeptin Serine/cysteine50mg/11ml water10–100m MPMSF(phenylmethylsulfonylfluoride)Serine120mg/ml water0.1–1.0m MAEBSF(4-[2-aminoethyl]benzenesulfonylfluoride hydrochloride)AEBSF is less toxicthan PMSFPepstatin A Aspartic5mg/7.3ml(1.0m M)1.0–10m Mmethanol or DMSOComplete tablets a Broad spectrum Tablets1X of multiple inhibitors Inhibitor cocktails I–VII b Broad spectrum100X1X of multiple inhibitors Inhibitor mixes Broad spectrum100X1X of multiple inhibitors B,FY,G,HP,M,P ca b c www.serva.de296Anthony C.Grabski debris and precipitated material by centrifugation or filtration.Since approximately50%of the volume of the insoluble residue will be trapped liquid,at least three volumes are required for greater than85%liquid recovery.Proteins are more stable at high concentration,but highly con-centrated extracts are difficult to process and aggregation can occur. Although a3:1ratio of disruption buffer to cell paste may be used to produce a more concentrated extract,5–10volumes of buffer are preferred and will yield more soluble protein and a less viscous extract.5.2.Cell disruption buffersBuffer B:50m M Tris/HCl(pH8.0),50m M NaCl,5%glycerol,0.5m M EDTA,0.5m M DTT or1.0m M THP,0.5m M benzamidine,1.0m M AEBSF.Buffer I:50m M sodium phosphate(pH7.5),150m M NaCl,5% glycerol,0.03%Brij35,0.5m M EDTA,0.5m M DTT or1.0m M THP, 1.0%Triton X-100,0.5m M benzamidine,1.0m M pepstatin A,1.0m M AEBSF.Alternatively,to avoid nuclear lysis in insect cells,substitute1.0% NP-40for Triton X-100.Buffer Y:50m M Tris/HCl(pH8.0),150m M LiCl,10%ethylene glycol,0.5m M EDTA,2.0m M THP,0.1%Triton X-100,0.5m M benzamidine,1.0m M E-64,1.0m M pepstatin A,1.0m M AEBSF.Note:Protease inhibitors may be omitted from or supplemented to (Table18.2)these buffers depending on the sensitivity of the protein target to proteolysis,and the degree or class of inhibition required.The ionic strength of buffers I and Y may need to be reduced or appropriate dilution of the extract performed if the initial protein fractionation step is ion-exchange chromatography.5.3.Microscale protocols for E.coli cell lysis5.3.1.Detergent-based reagent lysisLysis reagent recipe for10ml solution:10ml B-Per or BugBuster,20m l Lysonase Bioprocessing Reagent.Additives such as EDTA,protease inhi-bitors,5–10%glycerol,and reducing agents may be included in the lysis reagent as required to limit proteolysis and improve target protein solubility and stability.Downstream purification and analysis requirements must be considered when selecting lysis buffer components and their concentrations.1.Culture and express the target proteins using 1.0ml medium in2mlÂ96or5ml medium in10ml24-deep well plates with air permeable sealing film or BugStopper TM cap mats.2.Pellet the cells by centrifugation.3.Remove and discard the spent medium by aspiration.。