John A L Short Project Summary
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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
John A L Short Project Summary
- 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.