RecombinantExpressionandPurificationofE.

colihexosaminidase

LisaLi
CHM377

Abstract
Inthemureinrecyclingprocess, Nacetylglucosaminidase(NagZ)activity
degrades the anhydrodisaccharides (GlcNAc1,6anhydromuropeptides) into
monosaccharidesinthecytosolicstepsofthemuropeptiderecyclingpathway.Inaddition
toitsinvolvementinthebacterialmureinrecycling,theproductsofNagZactivityis
involvedwiththeinductionofampC lactamaseexpression.TheinhibitionofNagZ
activitymayinactivateampC lactamaseexpressionandsuggesttheabilitytoremove
antibioticresistance.Here,fivedifferentE.colistrains(BL21DE3,BL21DE3pLysS,
C43,Origami,Rosetta)previouslytransformedwithplasmidpET28b(+)wereculturedto
assessoptimalcellgrowthconditionsat30Cand37C.SDSPAGEgelelectrophoresis
andCoomassieBlueR250stainingwereusedforconfirmationofnagZoverexpression.
E.coliBL21DE3cellswerethenculturedforuseinproteinexpressionanalysis,purified
and visually confirmed through SDSPAGE and Coomassie Blue R250 staining. By
successfully overexpressing nagZ in BL21DE3 cells, we can eventually analyze the
activityofnagZquantitativelythroughitsenzymekinetics.

Introduction Murein sacculus turnover involves

Peptidoglycan or murein is a polymer
consisting of glycan strands and an
alternating sequence of N
acetylmuramicacid(aMurNAc)andN
acetylglucosamine (GlcNAc) linked by
1,4glycosidicbonds[2].Inbacteria,
peptidoglycan serves to maintain the
integrity of the cells infrastructure by
providingstructuralsupportforthecell.
Inmanycases,itcounteractstheeffects
ofosmoticpressure.Asthecellgrows,
theenlargementofthisstructurerequires
the synthesis of its monomers and its
integration into the existing murein.
During the murein tripeptide recycling
process,oldmaterialfromthebacterial
exoskeleton or sacculus is released as
turnoverproductsasthepolymergrows
bytheinsertionofnewglycanstrands.
enzymessuchaslytictransglycosylases
thatdegradeglycanstrandstoanhydro
disaccharides (GlcNAc1,6
anhydromuropeptides), endopeptidases
the cleave peptide rosslinks, and N
acetylmuramylLalanine amidases that
release oligopeptides from the murein
[1,3,4].
Anhydrodisaccharides
(GlcNAc1,6anhydromuropeptides)
formed from enzymatic cleavage are
thentransportedintothecytoplasmvia
AmpG permease. N
acetylglucosaminidase(NagZ)isneeded
for the degradation of the anhydro
disaccharides (GlcNAc1,6
anhydromuropeptides) into
monosaccharides in the cytosolic steps
of the muropeptide recycling pathway.

1
The GlcNAc1,6anhydromuropeptides
are substrates for NagZ and Nacetyl
muramylLalanine amidase. The
productsofNacetylmuramylLalanine
activity, GlcNAc-1,6-anhydro-MurNAc,
are then further processed into UDP
MurNAc pentapeptides that repress
ampC expression. The NagZprocessed
products, 1,6 anhydromuropeptides, act
to induce ampC lactamase. The
concentrations of UDP
MurNAcpentapeptide and GlcNAc1,6
anhydroMurNAc regulate AmpC
expression by binding to the
transcriptional activator, AmpR.
Therefore,theinactivationofNagZmay
inhibit the expression of lactamases
and keep bacteria in a sensitive state,
allowing resistant strains ofbacteria to
berenderedsensitive.[4]
NagZ is a member of the
glycosyl hydrolase (GH 3) family and
was first known as a cytoplasmic
enzyme active toward p-nitrophenyl--
N-acetyl-D-glucosaminide. In addition to
their primary hydrolytic activity, these
enzymes have also been shown to
catalyze transglycosylation reactions.
The structure of NagZ is the (/)8-
barrel structure of the catalytic domain.
The active site contains a pair of
catalytic residues Asp-Glu. The enzymes
is involved in substrate-assisted
catalysis, where catalytic glutamate acts
as a proton donor and the substrates 2-
acetamido moiety acts as a nucleophile,
forming an oxazoline reaction
intermediate. [5]
Methods
OptimizationofCellGrowthConditions
E. coli expression strains BL21DE3,
Origami, BL21DE3 pLysS, C43, and
Rosetta(DE3)wereassessedforoptimal
growth conditions. One colony from
eachtransformedstrainwasplacedinto
10 mL of LB containing 50 g/ml
kanamycin. 34 g/ml chloramphenicol
was added, in addition, for Rosetta
(DE3)andBL21DE3pLysScells.Cell
cultures weregrownovernightat37C
at225rpm.
Expression
Five250mlflasks containing100mlof
LBmedia(pH7.0)and3g/Lofglucose
were inoculated with 1ml of overnight
culture.Culturesweregrownat37Cat
225rpmuntilavalueofOD600reached
~0.40.Aliquotsof100Lofculturewere
extracted,placedintoamicrocentrifuge
tube,centrifugedat4000rpmfor5min,
and enriched for cells. The samples
servedas
controlsforcellsbeforeinducingprotein
expression. The remaining culture was
placedintotwo10mLculturesin50mL
centrifugetubesandinducedwithIPTG
toafinalconcentrationof1mMIPTG.
The10mLcultureswerethenplacedat
22Cand37Cat225rpm.Aliquotsof
100 Lwereharvestedatvarioustime
points (2 h, 4 h, and overnight),
centrifugedat4000rpmfor5minand
cell pellets were collected. Cell pellets
were stored at 20C until later use.
Remaining cell cultures were treated
with10%bleachanddiscarded.
SDSPAGE
10 mL of resolving gel was prepared
with2.5mlseparatinggelbuffer(1.5M
TrisClpH8.8),3.75ml(40%),100L
10% SDS, 3.6mL water, 50 L 10%
ammonium persulfate (APS), and 5L
TEMED.10mLof4%stackinggelwas
prepared with 6.4 mL water, 2.5 mL
stacking gel buffer (0.5 M TrisCl pH
2
6.8),100L10%SDS,50L10%APS,
and 10 L TEMED. Running buffer
(1X) was prepared with 0.025 M Tris,
0.192 M glycine, 0.1% SDS, pH 8.3
stored at 4C. 2X Laemmli sample
loading buffer was prepared with 62.5
mM TrisHCl, pH 6.8, 2% SDS, 25%
glycerol,0.01%BromophenolBlue,5%
2mercaptoethanol and diluted in a1:2
sampletobufferratiowithwatertoyield
2Xsampleloadingbuffer.
Cellpelletswereresuspendedin
40 L of 2X Laemmli sample loading
buffer,placedinasonicationbathfor3
min.andheatedfor5minona90Cdry
bath.Sampleswerethencentrifugedat
full speed for 1 min. SDSPAGE gels
previouslypreparedwereloadedwith12
Lofsamplesperwell.4LofaBroad
Range, 2212 kDa marker (New
EnglangdBioLabs)wasloadedforevery
setofsamples.Gelwassettorunat120
Vandincreasedto180Vaftersamples
ranpastthestackinggel.
CoomassieBlueR250Staining
After SDSPAGE was completed,
stackinggelswerecutandtheremaining
gelswerewashedwithwater.Thegels
wereplacedintoastainingcontainerand
stainedwith0.1%CoomassieBlueR250
in10%aceticacid,40%methanol,and
50%water.The stainingcontainerwas
heatedinamicrowaveovenfor45s,and
placedontorockerfor10min.Gelswere
destained by addition of water and
heatinginamicrowaveovenfor10min.
Purificationofrecombinantprotein
A colony of BL21(DE3) cells was
transformed with pET28nagZ and
cultured overnight with 5 mL LB
mediumsupplementedwith0.5mg/mL
kanamycin.Twoovernightcultureseach
were used to inoculate 3 2 L flasks
containing 0.5 L LBkanamycin. Cells
weregrownat37Cat250rpmuntilan
OD600valueof0.4wasobtained.Cells
were then induced with 0.5 mM IPTG
andincubatedat37Cfor4hours.Cells
were split into 6 centrifuge flasks and
harvestedthroughcentrifugationat5000
rpmfor20min.
Cellpelletswereresuspendedin
5mLoflysisbuffer(10mMTrisHCl,
50mMNaCl,10mMMgCl2,5mM
mercaptoethanol, 100 M
phenylmethylsulfonyl fluoride, 10
g/mLDNase,0.5mg/mllysozyme,pH
7.3), vortexed, and kept on ice. Cells
werelysedbysonification45timesfor
15 seconds followed by 45 seconds of
coolingonice.Samplesweretransferred
toacentrifugetubeandcentrifugedfor
20minat17,000rpminaSorvall5plus
centrifuge sing a SS34 rotor.
Supernatant was then transferred to a
cleancentrifugetube.Aliquotsof20L
fromthesupernatantweretransferredto
an Eppendorf tube for SDSPAGE
analysis.
0.5 mL TALON Co2+ columns
containing 1 mL of slurry were pre
equilibrated by passing five column
volumes(2.5mL)ofwaterfollowedby
2.5mLofequilibrationbuffer(10mM
TRISHCl,50mMNaCl,10mMMgCl2,
5mM mercaptoethanol,pH7.3)over
thecolumn.Supernatantwasloadedonto
the column and flowthrough was
collected. Aliquots of 20 L were
transferred to an Eppendorf tube for
SDSPAGE analysis. The column was
washedwith5mLofwashbuffer(10
mM TRISHCl,50 mM NaCl, 10 mM
MgCl2, 5mM mercaptoethanol,2mM
imidazole, pH 7.3). The wash was
collected and aliquots of 20 L to an
3
EppendorftubeforSDSPAGEanalysis.
ForelutionofthenagZprotein,2.5mL
ofelutionbuffer(10mMTrisHCl,50
mMNaCl,10mMMgClMgCl2, 5mM
mercaptoethanol, 50 mM imidazole,
pH 7.3) was loaded onto the column.
Theeluatewascollectedandanaliquot
of 20 L was transferred to an
EppendorftubeforSDSPAGEanalysis.

SDSPAGE:ProteinPurification
20Lof2xsampleloadingbufferwas
added to each sample followed by
heating to 90C on a heat block for 5
min.Sampleswerecentrifugedfor5min
atfullspeedand15Lofeachsample
wasloadedtothegel.5LofaBroad
Range,2212kDamarker(NewEngland
BioLabs)wasloadedontoeachgel.The
gel was then stained using Coomassie
brilliant blue according to methods
mentionedabove.

Results
OptimizationofCellGrowthConditions
OD600valuesofcellculturesforeach
strain were monitored at various time
points

4
 Strain 0h 2h 2.5h 3.0h 4.0h
BL21DE3 0.02 0.30 0.48
BL21DE3pLysS 0.03 0.21 0.46
C43 0.02 0.49
Origami 0.03 0.14 0.33 0.40
Rosetta(DE3) 0.03 0.08 0.16
Fig1.OD600valuesforcellgrowthofE.colistrains.Cellculturesweregrownat37Cat2250.41
rpmuntilavalueofOD600reached~0.40.

(Fig.1). When OD600 values of ~0.4
werereached,aliquots of100 Lwere
extracted and enriched for cells. SDS
PAGE gel electrophoresis was
performedtodetermine
the strain and cell growth conditions
(temperature and time) to be used for
cell culture for recombinant protein in
latersteps(Fig.2)

showedthegreatestexpressionofnagZof
allstrains.

From the Coomassie gel, BL21

(DE3)cellsshowedthegreatestamount
BL21DE3 Origami
ofnagZexpressionandwaschosentobe
usedfortransformationinthenextstep.
Other strains also showed increased
nagZ expression corresponding to the
bandsat~40kDa.

BL21DE3pLysS Rosetta(DE3) PurificationofRecombinantProtein

C43
Fig.2CoomassieBlueR250Stains.E.coli
strainswereanalyzedbasedoncellgrowth
conditions. Lanes for each set of strains
corespondtocellsgrownat30Cat2h,4h,
6h,respectively,and37Cat2h,4h,6h,
respectively. Bands at ~40 kDa marked
increasednagZexpression.BL21DE3cells
Aliquots of 20 L of flowthrough,
wash, and eluate were collected from
TALON Co2+ columns during protein
purification. Samples were used to
confirmsuccessfulpurificationofNagZ
via SDSPAGE gel electrophoresis.
Number of bands was observed to
decrease from preinduction cells to
flowthroughtowashandfinally,eluate.
This is consistent, given that the pre
inductioncells,flowthrough,andwash
shouldcontainsignificantlymorebands
than the eluate. Each eluate sample in
indicated in Fig. 3 and shows a clear
bandat~40kDacorrespondingtoNagZ
protein.Otherbandsseeninsampleof
eluate were artifacts, resulting most

5
likelyfrominsufficientwashing.Sample
5wasunabletobe
successfully purified. This may have
beenaresultofinsufficientwashingor
contamination between flowthrough,
washandeluatesolutions.
Sample1 Sample2
Sample3 Sample4
Sample5
Fig.3SDSPAGEgelelectrophoresisanalysisof
protein purification. 20 L samples of pre
induction cells, flowthrough, wash, and eluate
from each strain were collected during protein
purification.Greenarrowsindicatepreinduction
samples. Blue arrows indicate flowthrough
samples. Yellow arrows indicate samples
containing wash. Red arrow indicates sample
containingpurifiedprotein.
Discussion
Expression of nagZ was induced into
five E. coli strains (BL21DE3, BL21
DE3pLysS,C43,Origami,andRosetta
(DE3)) to determine the strain that
wouldoverexpressnagZtothegreatest
degree. Furthermore, expression trials
allowedfortheexaminationofoptimal
cell growth conditions for nagZ
expression,intermsoftemperatureand
time. Our SDSPAGE results indicated
thatBL21DE3containedasignificantly
greater quantity ofNagZ protein while
Origami showed weak signs of
overexpressionofNagZ.At 2hoursand
22 degrees, BL21DE3 showed the
greatest amount of nagZ expression.
Therefore, BL21DE3 was chosen to
perform the next steps of purification
and confirmation with SDSPAGE
analysis.
To purify the protein, a crude
lysate was obtained and purified via a
TALONCo2+column.Afterpurification,
samplespreviouslytakenfromtheflow
through, wash, and eluate were
compared to ensure successful
purification.SDSPAGEwasperformed
forthispurpose.AccordingtoFig.3,the
bandsatkDarepresentingnagZhadless
bands surroundingitintheeluatethan
thewash,whichwas hadless thanthe
flowthrough. Therefore, protein
purification was successful with the
exception of a few samples containing
artifacts that resulted most likely from
insufficientwashing.
BybeingabletopurifynagZ,we
will be able to study its kinetic
properties.Thegoal,forthenextstep,is
tostudyhowenzymaticactivityofNagZ
isaffectedbysubstrateconcentration.
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