Expression and Purification of UDP-N-acetylenolpyruvoylglucosamine Reductase (MurB)

Expression and Purification of a novel recombinant form of UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) for structural and functional studies

Abstract:

A new variant of the plasmid construct designed to express the protein from Enterococcus faecalis in E.coli was investigated. To test this new construct, the quality and yield of the protein was evaluated in a form suitable for further structural studies. UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) was the enzyme of interest; thus, it was cloned into a pProExa expression vector. An N-terminal polyhistidine tag was added to the vector to enable purification using immobilised metal affinity chromatography (IMAC). The construct was also engineered to contain a Tobacco etch Virus (TEV) protease site, which later allowed for His tag cleavage preventing any interference with downstream activity and analysis. Concentration of the samples taken throughout the purification were calculated using a standard Bradford Test. SDS-PAGE analysis was carried out providing a visual representation of protein purification as a decreased number of minor bands were present in the later stages of purification. Finally, a Western Blot was carried out to confirm the presence of MurB and, once more, providing proof of purification as the band intensity increased further on in the purification process.

1. Introduction

Enterococcus faecalis

E.faecalis is a gram-positive opportunistic bacterium found to be the etiological agent of many postsurgical infections. It most commonly colonizes in the bladder and kidneys leading to urinary tract infections. E.faecalis is also regularly found to be the causative agent of endocarditis, secondary bacteremia, food poisoning and inflammatory bowel disease(1). These infections can be challenging to treat since many E.faecalis isolates possess antibiotic resistance to gentamicin, ampicillin and vancomycin(2). Patients suffering with an enterococcal infection may have a extensive stay in hospital due to the persistence of the infection. If anything healthcare settings have provided a platform for the bacteria to thrive and evolve under the selective pressures of a competitive environment. Many enterococci have developed genetic strategies of avoiding antibiotics such as; modification of drug targets, inactivation of therapeutic agents, overexpression of efflux pumps and a cell envelope adaptive response promoting survival in the human host(3).

However, there is hope to fight infections caused by E.faecalis by designing a new generation of inhibitors that can prevent cell wall formation leading to apoptosis.

Role of Mur enzymes in peptidoglycan biosynthesis

Oxidoreductases and ligases are essential for bacterial wall biosynthesis. There are 6 enzymes involved in the transformations required to produce the peptidoglycan precursor which then goes on to form the bacterial cell wall. MurA and MurB synthesize UDP-MurNAc from UDP-GlcNAc. This enol ether transfer and reduction step produces a lactyl ether bridge that connects the repeating disaccharide and pentapeptide units that form the peptidoglycan layer giving the cell wall rigidity(4). This reaction is followed by the action of four ligases, MurC, MurD, MurE and MurF, retrospectively adding amino acids to UDP-MurNAc, producing the UDP-MurNAc-pentapeptide. Peptidoglycan biosynthesis is necessary for bacterial cell survival; prevention leads to disrupted cell wall assembly resulting in bacterial cell lysis. Therefore, making the study of these enzymes structure and function attractive in novel antibacterial drug design.

Biochemistry, inhibitors and structure of MurB

The aim of this study is to purify UDP-N-acetylenolpyruvylglucosamine reductase, MurB, from a new variant of the plasmid construct from Enterococcus faecalis in E.coli. MurB enzymes have been previously purified from Escherichia coli, S. aureus, and Streptococcus pneumoniae allowing for structure-based approaches to design inhibitors.

MurB is a mixed αβ protein composed of three domains. Substrate binding leads to a conformational change where domain 3 undergoes a rigid-body rotation away from domain 1 and 2 resulting in closure of the substrate-binding channel

MurB catalyses the reduction of UDP-N-acetylglucosamine enolpyruvate to UDP-N-acetylmuramic acid in two half-reactions. FAD in the active site is reduced by NADPH, then electrons are transferred to enolpyruvyl group. Steady-state kinetic studies show NADPH and UDP-N-acetylglucosamine enolpyruvate work via a ping pong bi bi double competitive substrate inhibition mechanism (5) This suggests that both substrates share a sub-site meaning they can’t both be bound to MurB at once.

The monomeric enzyme has a characteristic flavin adenine dinucleotide, FAD, binding domain involved in the first stage of the reaction. This FAD cofactor mediates hydride transfer from the NADPH and enolpyruvyl during catalysis(6).

It has been found that the Arg159 and Glu325 residues are responsible for transition state stabilisation by interacting with the carboxylate of the substrate. It has also been suggested that Tyr190, Lys217, Asn233, and Glu288 residues interact with diphosphate moieties in the substrate.

In the second half of the reaction UDP-N-acetylglucosamine enolpyruvate induces changes, in domain 3, that disrupt the interaction between Tyr190 and Tyr250 at the side of the channel leading to the active site. Tyr190 moves over the channel opening forming a hydrogen bond with an oxygen on the substrate resulting in closure of the channel over the substrate (7) These types of insights into MurB’s binding strategies and mechanisms are important steps in the  development of targets for antibacterial chemotherapy.

The diphosphate interactions between enzyme and substrate have been simulated using 4-thiazolidinone. This novel inhibitor occupies the disulphide moiety moving the side chains in a similar way to UDP-N-acetylglucosamine enolpyruvate preventing its binding. Therefore, preventing the synthesis of UDP-N-acetylmuramic acid.

2.0. Materials and Methods No more than 500 words in total for this section

2.1 Transformation and Overexpression of E. faecalis MurB

The gene for E. faecalis MurB was cloned into the E. coli expression vector pProEx (Invitrogen) such that the gene was placed under transcriptional control of the plasmid encoded Tac promotor and was used to an N-terminal poly-histidine and Tobacco Etch Virus (TEV) protease site.  The E. coli protein expression strain BL21(DE3)Star:pROSETTA cells were transformed with the MurB expression vector and grown in 2YT auto induction media (Formedia). Cells were grown at 37˚C and 180 rpm for 5 hours before reducing the temperature to 18 ˚C at the same speed for a further 12 hours of growth.

2.2 Purification of E. faecalis MurB

Following growth, cells were harvested by centrifugation at 8000rpm for 10 minutes. Following removal of the supernatant; cells were resuspended in a (50mM Na₂PO₄ pH 7.5) buffer and 250µl of lysozyme. The cells were then disrupted using sonication allowing release of the plasmid, and subsequently centrifuged at 20,000rpm for 30 minutes at 4^oC. The supernatant containing MurB was collected.

MurB was then purified using Immobilised Metal Affinity Chromatography (IMAC). The chromatography column was charged by binding Nickel (using 20ml of 50mM Sodium Acetate, pH 4.0, 10mM NiCl₂)to a Chelating Sephorase chromatography resin. Buffer A (10mM Imidazole pH 7.0) was added allowing for equilibration of the column. The supernatant collected in the previous step was added to the column and left to elute from the column before adding buffer A and collecting 5mls of the flow through to gather the unbound protein.

Following this, washing steps took place with buffers (B and C) of increasing concentrations of imidazole (25mM and 500mM) to compete with the Histidine binding to the Nickle causing the protein to detach. Different fractions of MurB were collected and used in SDS-PAGE analysis. All buffers had a pH of 7.0 to deprotonate the Histidine tags allowing them to bind to the Nickel.

TEV protease was used to cleave the histidine tag preventing any inference with downstream activity in the SDS-PAGE. The pooled IMAC fractions produced from washing with buffer C were placed into dialysis tubing before being left in a 50mM NaHPO₄ pH 7.5, 100mM NaCl buffer overnight. Allowing for the imidazole to outcompete the MurB for His tag binding.

Dialysed protein was applied to the Ni column allowing separation of any un-cleaved protein including the TEV. The column was then washed with buffer A (10mM Imidazole) eluting out the MurB successfully cleaved by TEV protease. Any remaining MurB still attached to TEV was washed out of the column using buffer C (500mM Imidazole).

Protein concentration of MurB in each sample was determined using the Bradford test.

Samples were collect throughout the purification process and diluted as shown in supplementary table 1 (see supplementary data). 40µls of each sample were loaded as shown in table 2A (see supplementary data) to produce and SDS-PAGE. Gels were run at 200V for 45 minutes, then stained using an Instant Blue Coomassie protein stain.

2.3 Western Blot

10µl of the same samples as above were loaded into a gel, as shown in supplementary table 2B (see supplementary data), and then blotted onto a PDVF membrane. The membrane was then probed by a monoclonal antibody specific for the histidine tag on the MurB. The antibody also allowed for band visualisation on the gel due to the Horse radish peroxidase (HRP) enzyme that catalyses a reaction emitting low intensity light at 428nm.

3. Results  

First a standardises Bradford test was carried out using known protein concentrations to produce a standard curve shown in Supplementary Figure 1. A straight-line correlation was indicated producing a standard equation of y=0.0582x. This was then used to determine the concentration of protein collected in the samples as shown in Supplementary Table 1.

The crude extract and unbound protein flow through (UPFT) both had high protein concentrations of 7.6µg/µl and 7.1µg/µl retrospectively (see Supplementary Table 3A). High concentrations are expected since the crude extract contained a mixture of proteins that would have been released by breaking open of the cell this is also shown by the vast number of minor bands that can be seen in lane 2 of the gel (Figure 1). Similarly, the UPFT sample in lane 3 shows an assortment of bands, however the band around 52kD is less pronounced showing that some of the MurB bound to the chromatography column, therefore was not present in the flow through as expected. The mass of MurB is only 41.9kD(8) however the reason for the higher mass shown in Figure 1. SDS-PAGE of Enterococcus faecalis MurB protein purification using Immobilised Metal Affinity Chromatography (IMAC). A major band around 52kDa corresponds to MurB, which according to sequence has a molecular weight of 41.9kDa. Lanes from left to right: Marker (lane 1), Crude extract (lane 2), Unbound protein flow through (Lane 3), B1 (Lane 4), B2 (Lane 5), C1 (Lane 6), C2 (Lane 7), Pooled IMAC fractions (Lane 8), TEV low-I Elution (Lane 9) and TEV high-I Elution (Lane 10). 

the gel is due the attachment of the histidine tag on the N-terminal peptide and the addition of TEV.

The concentration of protein eluted from the IMAC column increased from 1.79µg/µl in sample B1 to 2.28µg/µl in sample C1 as the imidazole concentration in the buffer increased from 25mM to 500mM. This is because the imidazole can compete with the his-tag for Ni binding leading to elution of MurB from the column. The increase of MurB present in the samples can also be seen by contrast of band density in lanes 4 and 5 compared to lanes 6 and 7.

All of the C fractions, washed with 500mM of imidazole, were pooled together. Concentration of the combined fractions was determined as 1.41µg/µl. The corresponding band should have been visible in lane 8 of the SDS-PAGE however no band can be seen. This was not expected and most likely due to errors in loading the sample.

Excess imidazole may cause the His tags to aggregate resulting in interference with downstream activity and was therefore removed by Figure 2. Western blot of Enterococcus faecalis MurB protein purification using Immobilised Metal Affinity Chromatography (IMAC). Lanes from left to right: His-tagged Protein Standard Marker (lane 1), Crude extract (lane 2), Unbound protein flow through (Lane 3), B1 (Lane 4), B2 (Lane 5), C1 (Lane 6), C2 (Lane 7), Pooled IMAC fractions (Lane 8), TEV low-I Elution (Lane 9) and TEV high-I Elution (Lane 10). 

dialysis, whilst the histidine tag was being cleaved. Following dialysis, protein concentration increased to 10.33 µg/µl.

The MurB protein was re-purified by adding the dialysed protein and buffer A to the column. Following this a protein concentration of 5.56µg/µl was determined. The MurB collected in this sample would have been free of TEV showing that the TEV protease cleavage was successful.

The next sample was produced by washing with buffer C. The higher concentration of imidazole would have led to the remaining MurB, still attached to his-tags, to elute from the column. A protein concentration of 4.60µg/µl was calculated. Both the TEV low-I elution and TEV high-I elution had a high concentration of purified MurB. This can be seen in Figure 1 showing thick bands around 52kDa representing the high concentration, also the lack of other bands in the lane show that the sample was pure.

The SDS-PAGE allowed separation of protein based on molecular weight. The next step provided conformation that the protein present was in fact MurB using specific antibodies on a western blot. A monoclonal antibody specific to the histidine tag on MurB, as well as having a Horse radish peroxidase (HPR) enzyme attached, was used. In figure 2 it can clearly be seen that the band intensities increase in lanes 6,7,9 and 10 as MurB concentration increases. Again, a band is missing in lane 8, it seems unlikely that loading errors were made twice for the same sample. However, it may be more plausible that the sample was mixed up with something else and labelled as “pooled IMAC fractions”.

Finally, the total protein in the crude extract, dialysed IMAC, TEV low-I elution and TEV high-I elution were calculated to be 175.29mg, 154.89mg, 104.73mg and 50.15mg retrospectively.  Meaning that TEV protease successfully cleaved 68% of the sample. From the total protein it can also be determined that the final recovery yield for purified MurB was 104.73mg/2L culture.

4. Discussion No more than 250 words and should include suggestions for improvement of the experiment

The most significant error in this experiment was the absence of a band in lane 8, representing the pooled IMAC fractions, in both the SDS-PAGE and the western blot.

Theoretically the use of an E.coli expression vector should have expressed a high protein yield.

In order to confidently state that MurB can be purified from Enterococcus faecalis in E.coli repeats are needed.

22.5 units should have been enough to cleave 154µg of protein overnight. The reason that it didn’t may have been due to the substrate tested on was different so acted at a slightly higher rate. Or it may be possible that some of the enzyme had become denatured so there was a decrease in overall specific activity. TEV protease reaches its optimum at a pH of 8 and 30^oC however the cleavage conditions were a pH of 7.5 set at room temperature. This may explain why only 68% of protein was cleaved rather than the possible 85%.

Currently MurA is the only enzyme of the Mur family with a known inhibitor, fosfomycin. As previously discussed, 4-Thiazolidinone compounds have been designed as MurB inhibitors(5). However, there is ongoing research in the area of Mur enzymes. Having cheap and easy methods of purifying MurB will mean large scale research can be carried out on designing novel bacterial cell wall inhibitors.

References

Supplementary Data

Supplementary Figure 1. Standard curve produced using BSA. Bradford reagent was used to develop colour which was read at an absorbance of OD₅₉₅ in a spectrophotometer.

Supplementary Table 1. Sample preparation for SDS-PAGE

Supplementary Table 2. (A) Loading of lanes in SDS-PAGE (40µl samples). (B) Loading of lanes in SDS-PAGE (10µl samples) in preparation for Western Blot

Supplementary Table 3. Determination of protein concentration throughout the purification process using y=0.0582x. (A) calculated protein concentration using Concentration (µg/µl)=0.0582/Absorbance (595nm). (B) Calculation of the total protein concentration in Crude, Dialysed IMAC, TEV-Low and TEV-High fractions using concentration from A and measured volumes.

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