The project aimed to clone, express, and purify E. coli Ddlb enzyme to use in further experiments exploring its potential as an antibiotic target. Key steps included: 1. Amplifying the Ddlb gene from E. coli via PCR and cloning it into a pET-15b expression vector. 2. Transforming E. coli with the expression construct and inducing expression, which successfully produced the Ddlb protein. 3. Purifying the expressed Ddlb protein via native batch purification using Talon resin, which isolated Ddlb with only one contaminant. 4. Removing the His-tag from the purified protein to prepare it for future inhibitor assays and crystall

1. Project Title:
The Cloning, Expression and Purification of E. coli Ddlb
Project Supervisor: Dr Julieanne Bostock
Background and Aims
Peptidoglycan is a bacterial polymer surrounding the bacterial plasma membrane. It is an essential
component of the bacterial cell wall providing the structural integrity necessary to resist internal
osmotic pressure and to prevent cell lysis (Isono et al., 1985; Kimura et al., 1998). It is synthesised by
a series of enzymes which are inhibited by several established antibiotics as shown in Figure 1.
PEP
NADPH
L-Ala
D-Glu
A2pm or Lys
D-ala-D-ala
Undecaprenyl-P
UDP-GlcNAc
vancomycin
bacitracin
moenomycin
ß-lactams
MurA
MurB
MurC
MurD
MurE
MurF
MraY
MurG
2 D-alaDdl
cycloserine
L-ala
Alr
cycloserine
L-Glu
MurI
fosfomycin
UDP-GlcNAc
UDP-GlcNAc-enolpyruvate
UDP-MurNAc
UDP-MurNAc-L-Ala
UDP-MurNAc-dipeptide
UDP-MurNAc-tripeptide
UDP-MurNAc-pentapeptide
Lipid I
Lipid II
Nascent peptidoglycan + Undecaprenyl-P-P
Cross-linking to pre-existing Peptidoglycan
PEP
NADPH
L-Ala
D-Glu
A2pm or Lys
D-ala-D-ala
Undecaprenyl-P
UDP-GlcNAc
vancomycin
bacitracin
moenomycin
ß-lactams
MurA
MurB
MurC
MurD
MurE
MurF
MraY
MurG
2 D-alaDdl
cycloserine
L-ala
Alr
cycloserine
L-Glu
MurI
fosfomycin
Figure 1: Bacterial Peptidoglycan biosynthesis and inhibition by established antibiotics;
substrates in blue, inhibitors in red
However, emerging drug resistance is challenging the clinical effectiveness of these antibiotics, where
new solutions are urgently required. (Levy 1998). The bacterial enzyme D-alanine-D-alanine ligase
(Ddlb) is a relatively unexploited drug target synthesising an essential D alanine – Danyl dipeptide unit
present in the cell wall (Besong et al., 2005; Ellsworth et al., 1996; Isono et al., 1985; Kimura et al.,
1998) Its potential as an antibiotic target has already been established as it is inhibited by D-
cycloserine, a broad spectrum antibiotic which is a structural analogue of D-alanine ( Neuhouse and
Lynch., 1964). However, toxicity and adverse side effects have limited the utility of cycloserine as an
antibiotic and is presently not widely used in the clinic.
The aim of the project is therefore to clone, express and purify E. coli Ddlb, to be used in further
experiments. Ddlb has already been crystallised (Fan et al., 1994), and potential inhibitors have
been created using computer simulations (Besong et al., 2005) but these need to be practically tested
as the simulations may not reflect all of the molecular interactions in the enzyme inhibitor complex.
The purified enzyme will in the future be crystallised with various inhibitors, in order to see the specific
molecular interactions between the enzyme and the inhibitor. As a result existing inhibitors could be
modified or novel ones produced in order to develop effective inhibitors of Ddlb for commercial and
clinical usage.

2. Expression
Technique Rationale
Transformation of E. coli B121 Star
Expression strain with Bostock Agenda
from step 10 (with amphicillin for selection)
The B121 Star strain of E. coli is geared towards high expression of induced plasmid i.e. Ddlb
Expression in LB broth (with Carbenicillin
for selection) , inducement at 0.6 OD600
with IPTG
Expression of Ddlb enzyme
Strategy and Results
Cloning
Technique Rationale Result
Touchdown Polymerase Chain Reaction using KOD and
Extensor and Thermoprime DNA Polymerases
To determine which enzyme is most appropriate to amplify E. coli Ddlb gene.
DNA AGE
To check that the PCR has worked in amplifying
the right gene.
The Extensor polymerase produced the
gene of the correct size 1000 bp.
DNA Agarose Gel Extraction using QIAEX II To extract and purify Ddlb
DNA AGE of pET15b and purified Ddlb
To determine quantity of Ddlb and pET-15b for
restriction Digestion The vector used was pET-
15b, which has the antibiotic marker amphicillin
for selection.
Ddlb = 100ng per band
pET15B = 55ng per band
Restriction Enzyme Digestion of pet15b and Ddlb with
Nde I and Bam HI (RED)
To prepare vector and insert for ligation to make recombinant construct of Ddlb and pET-
15B (Bostock Agenda)
Mini-Elute PCR Purification Kit Protocol To purify restriction enzyme digestion products.
DBA Ligase Reaction
To ligate the Ddlb and pET-15b together to produce the recombinant construct (Bostock
Agenda).
E. coli Transformation using XLI-Blue Supercompetant
cells
To transform E. coli with Bostock Agenda for expression later.
Wizard Plus Minipreps DNA purification system; RED;
DNA AGE
Screening for Successful uptake of Bostock
Agenda in E.coli
Four of the 10 colonies examined had
successful uptake of Bostock Agenda
RED; DNA AGE
To screen for correct orientation of Ddlb in
Bostock Agenda
The insert was in the correct orientation
in all four of the colonies

3. Purification
Technique Rationale Result
Protein Extraction using Bug Buster
Protocol
To extract Ddlb enzyme from E.coli cells
SDS PAGE of extracted protein
To determine if protein has been
successfully extracted
The protein was successfully extracted where there was a
large yield of expected protein at 35 kDa but contained other
proteins (as expected).
Trial 1 Trial 2
Technique Rationale Technique Rationale
Batch Protein
Purification under
Native conditions
using Ni-NTA resin;
SDS-PAGE
To purify the Ddlb
protein from all other
contaminates using
the His-tag to bind to
the resin.
Batch Purification
under native
conditions using
Talon Resin
(Cobalt);
SDS-PAGE
To purify the Ddlb
protein from all
other contaminates
using the His-tag to
bind to the resin.
The protein was successfully purified under both conditions.
However, the Talon resin was more specific where under the
Ni-NTA there was only one contaminating protein at 75kDa.
Therefore, for large scale purification Talon resin should be
used.
- -
Bradford Assay To determine the
concentration of
Ddlb prior to
dialysis.
The Ddlb concentration in Trial 2 was found to be 0.212
mg/ml
Dialysis of Protein using Slide – a lyzer
Dialysis cassettes
To exchange the elution buffer of Batch purification to one optimal for Ddlb
Bradford Assay
To determine concentration of protein
purified.
The Trial 1 Ddlb concentration was 1.6 mg/ml, whereas for
Trial 2 was 0.239 mg/ml. The Ddlb was 12.7% more
concentrated after dialysis in Trial 2.
Large scale
Removal of His-Tag
using Thrombin;
SDS-PAGE
This is the key
procedure to remove
the His-Tag for future
assays and for
crystallography.
Unfortunately due to time constraints
removal of His-Tag for Trial 2 was not
carried out.
The His-Tag was successfully removed where on the SDS-
PAGE gel the protein had a lower molecular weight than that
Ddlb with His-Tag still present.
Dialysis of Protein using Slide – a lyzer
Dialysis cassettes (Trial 1)
To exchange the elution buffer of thrombin removal to one optimal for Ddlb for Trail 1.
Bradford Assay (Trial 1)
To determine concentration of protein
after His-Tag removal
The Trial 1 Ddlb concentration was diluted down to 0.15
mg/ml, a decrease of 90%.
Ddlb activity assay (Trial 1 and Trial 2) To determine if the Ddlb is active For Trial 1 and Trial 2 the Ddlb was active.
IC50 Assay of Ddlb using D- cycloserine
(Trial 1)
To determine if Ddlb is working
effectively.
The IC50 of Ddlb is 2.399mM
Conclusion
The aim of the research proposal has been met with the enzyme Ddlb being successfully cloned,
expressed and purified from E.coli Ddlb. The protein can thus be assayed with various inhibitors and
be crystallised for future investigation.
Given more time, the removal of His-Tag from Trial 2 and its IC50 would have been carried out but
this was due to problems encountered in finding the optimal conditions for Ddlb to be active in.