2.2.1 Transformation and expression of PA2794 and a PA2794 F129A mutant
A recombinant plasmid based on pET15b was designed containing a T7 promoter site upstream of a number of sequences coding for restriction enzymes and a hexa-his tag coding sequence upstream of a thrombin cleavage site. To afford PA2794 with a N-terminal hexa-His tag, the plasmid was designed to incorporate the PA2794 gene sequence into the plasmid between NdeI and BamHI restriction enzyme sites using codons optimised for E. coli (Appendix 1). The resulting recombinant plasmid (purchased from GenScript) was transformed via heat shock into chemically competent E.coli BL21 DE3 cells and deemed successful by a display of ampicillin resistance during growth on LB agar.
Incorporation of the PA2794 recombinant plasmid did not appear to be detrimental to bacterial growth which was monitored prior to induction of the protein of interest. Although a protocol for expression and purification of PA2794 has previously been determined, incubation temperature and time after induction were investigated in order to optimise expression levels (Table 2.1). Prior to induction, a culture sample was taken from each condition and treated exactly the same but without the addition of IPTG to provide an indication of the expression levels of E. coli proteins. Expression levels were determined using SDS PAGE analysis of crude lysates from small scale grows up under these induction conditions (Figure 2.7).
Samples that had been induced with IPTG displayed an over-expressed protein between the marker 45.0 kDa and 66.2 kDa bands (Figure 2.7a) which was much less prominent in samples that had not been induced (Figure 2.7b). Therefore this band was tentatively assigned as corresponding to the desired protein PA2794 (47.1 kDa). There were a number of other significant proteins detected by SDS-PAGE, such as one at ~ 25 kDa, which were thought to potentially be truncated PA2794 protein and could require further investigation of expression conditions. However as these were also present in the non-induced samples they were assigned as highly expressed E. coli proteins and not over-expressed truncated PA2794 (Figure 2.7). Upon varying the concentration of IPTG there was no significant difference observed in the level of expression between induction with 0.1 mM and 0.5 mM IPTG. However, it was apparent that expression levels could be slightly increased by inducing at a temperature of 37 °C compared to 16 °C and by leaving the cells to grow for longer periods of time (Figure 2.7a). Therefore, in subsequent grow ups, expression conditions involved incubation of cells at 37 °C for 18 hrs following induction with IPTG.
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Table 2.1 Expression trial conditions to optimise production of PA2794.
Condition A B C D E F G H Post induction incubation time 4 hrs 18 hrs IPTG concentration 0.1 mM 0.5 mM 0.1 mM 0.5 mM Post induction incubation temp 16 °C 37 °C 16 °C 37 °C 16 °C 37 °C 16 °C 37 °C
Figure 2.7 SDS PAGE analysis of crude lysate samples of the PA2794 enzyme a) under different induction conditions after induction with 0.1 mM (A, B, E, F) or 0.5 mM (C, D, G, H) IPTG and b) under these conditions but with no induction with IPTG.
64 Mutation of the PA2794 phenylalanine 129 to an alanine residue was carried out on this recombinant plasmid to afford an enzyme that has previously been suggested to increase PA2794 activity with Neu5Ac (1.1) analogues. The forward primer was designed to replace the phenylalanine129 codon (TCC) with the most similar codon for an alanine (GCC). PCR utilising a Phusion site-directed mutagenesis kit was carried out with a number of different annealing temperatures (55 °C-72 °C) and the DNA amplification analysed on an acrylamide gel. The presence of a single band on the gel demonstrated successful amplification of a specific length of DNA. However the DNA band appeared slightly higher on the gel than expected under all conditions, suggesting the amplified DNA may have more base pairs than the desired recombinant plasmid (7.2 Kilobases) (Figure 2.8). As it was very unlikely that a larger piece of DNA had been amplified with no amplification of the desired plasmid, it was instead suggested that this was the correct stretch of DNA and the apparent increase in number of base pairs was attributed to analysis error. For example, it is hard to distinguish between the standards with large numbers of kilobases as the resolution between them is low. Additionally it was noted that the PCR samples were in a different buffer to that of the standards and hence this may affect their diffusion through the gel. In order to fully assess the success of the PCR reaction the DNA was extracted from the gel and transformed via heat shock into chemically competent E.coli BL21 DE3 cells.
Figure 2.8 DNA agarose gel following a point mutation PCR of PA2794 pEt15b recombinant plasmid.
The growth of these colonies on ampicillin containing LB agar suggested successful amplification and transformation of the recombinant plasmid and sequencing of a number of colonies (Appendix 2) showed incorporation of the F129A mutation in all cases. Prior to DNA extraction the individual colonies were cultured overnight (10 mL LB containing ampicillin 100 µg mL-1) and a small sample inoculated on LB agar containing ampicillin 100 µg mL-1 to ensure that colonies were available containing the DNA that had been sequenced.
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2.2.2 Purification of PA2794 and PA2794 F129A
Following induction of the desired enzyme (PA2794 and PA2794 F129A) in E. coli BL21 DE3 cells on a large scale (3 L LB), cell pellets were resuspended and lysed in Tris-HCl buffer (50 mM, pH 7.5), 10 % glycerol containing 10 mM imidazole, Benzonase (25 U/L grow up) and protease inhibitor tablet (used as instructed). Initial purification from other buffer soluble proteins was attempted by loading onto a Nickel affinity column and applying an increasing imidazole concentration gradient in Tris-HCl buffer (50 mM, pH 7.5) and 10 % glycerol. Presence of a UV Abs280 peak during washing with higher concentrations of imidazole (after loading and washing the column with low concentrations of imidazole) confirmed over-expression of the His6-tagged PA2794 protein (Appendix 3). The eluted protein corresponding to the predominant UV Abs280 peaks was confirmed as the desired PA2794 or PA2794 F129A enzyme by SDS-PAGE analysis which displayed over-expressed protein with the expected molecular weight (Figure 2.9a,b).
Figure 2.9 SDS PAGE analysis of a) PA2794 and b) PA2794 F129A after purification using a Nickel affinity column.
Although this method was successful in purifying PA2794 and PA2794 F129A from the majority of the other proteins, impurities are still evident in both samples, especially for enzyme collected at the lower concentrations of imidazole. It was proposed that in subsequent purifications this could be improved upon by increasing the gradient of imidazole less steeply or by introducing step-wise increments in imidazole concentration instead of application of a linear gradient. However size exclusion was utilised to improve the purity of the PA2794 and PA2794 F129A samples. Elution of the column in Tris-HCl buffer (50 mM, pH 7.5) and 10 % glycerol, resulted in
66 the desired highly pure PA2794, or PA2794 F129A, as observed by one symmetrical peak in the gel filtration UV trace (Appendix 4) and only one protein band in the SDS PAGE analysis of the fractions whereby an increase in Abs280 was detected (Figure 2.10a,b).
It was apparent from the SDS PAGE gels that considerably less PA2794 F129A enzyme was expressed compared to the native PA2794 enzyme. However satisfactory yields of 9 mg per L of LB grow up for PA2794 and 4 mg per L of LB culture for PA2794 F129A were extracted. Following purification, enzymes were concentrated to 3.5 mg mL-1 in Tris-HCl buffer (50 mM, pH 7.5), 10 % glycerol and flash frozen prior to storage at -80 °C.
Figure 2.10 SDS PAGE analysis of a) PA2794 and b) PA2794 F129A following purification with a size exclusion column (120 mL, HiLoad 16/600) after a Nickel affinity column.
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