Applications

• LysisofGram-negativeorGram-positivebacteria(Table1)forproteinpurification(Fig.1).
• Purificationofnucleicacidsfrombacteria.

Ready-Lyse™LysozymeSolutionisarecombinantlysozymepreparation,derivedfromanonmammalian,nonaviansource,thatisusefulforthelysisofGram-negative(suchasE.coli)andGram-positive(suchasBacillussp.)bacteria.ThespecificactivityofReady-LyseLysozymeis200-foldhigherthanthespecificactivityofegg-whitelysozymeand,therefore,lesslysozymeisneededinareaction.Also,unlikeegg-whitelysozyme,Ready-LyseLysozymeSolutionisstableat-20°C,eliminatingtheneedtoprepareafreshsolutionforeachuse.TheuseofReady-LyseLysozymeresultsinhigheryieldsofproteinthancanbeobtainedwithstandardeggwhitelysozyme.

UnitDefinition:OneunitofReady-LyseLysozymeproducesadecreaseinA₃₅₀of0.001perminuteat23°Cwitha0.5mg/mlsUSPensionoflyophilizedE.coliK802cellsin50mMTris-HCl(pH7.5).

StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,0.1mMEDTA,1mMDTT,and0.1%Triton®X-100.

Figure1.LysiswithReady-Lyse™Lysozymeincreasesyieldsofnucleicacids.500µgpermlofpHC79cosmidDNAwasincubatedfor15minutesat22°CinTEbuffer(25mMTris(pH8.0),10mMEDTA)containingeither5µg(30KU)permlofReady-LyseLysozymeor500µg(25KU)permlofeggwhitelysozyme.Thesolutionswerecentrifugedfor10minutesandthepelletswereresuspendedinTEbuffercontaining0.1%SDS.DNAinsupernatantsandpelletswasseparatedbyelectrophoresisina0.8%agarosegel.Approximately50%oftheDNAwaslostduetoprecipitationbyeggwhitelysozyme(EW),whileReady-LyseLysozyme(RL)causedminimalprecipitationlossesofDNAcomparedtocontrol(C)sampleswithoutlysozyme.

Figure2.UseofReady-Lyse™LysozymeSolutiontorecoverrecombinantproteins.OnemlofinducedcellsfromarecombinantE.coliclonewaspelletedbymicrocentrifugationbeforeinductionandat1and3hoursafterinduction.Eachsamplewasresuspendedin50µlofcoldTEBGbuffer.OneµlofReady-LyseSolutionwasaddedtoeachsuspensionandthecellswereincubatedatroomtemperaturefor30minutes.Thecelldebriswaspelletedand10µlofthesupernatantwererunonanSDS-PAGEgel.Lane1,molecularweightMarkers;Lanes2-4,timepointsofinduction.Theinducedproteinisdesignatedbyanarrow.