PAM and TycAS563A
Laboratory Objective #2
Produce and test PAM and TycA of Pathway 1
Figure 1. Schematic diagram representing the conjugation of PAM-SnoopTag (blue) and TycA-SpyTag (orange) enzymes to βPFD-SnoopCatcher (light red) and αPFD-SpyCatcher (dark red) via the catcher-tag system. PAM catalyses the conversion of ⍺-phenylalanine to 𝛽-phenylalanine. TycAS563A catalyses the production of phenylisoserinyl-CoA by the addition of the CoA thioester group to 𝛽-phenylalanine.
Our second lab objective was to run and test pathway 1. We aimed to produce and synthesise the reaction enzymes involved in the biosynthesis of Paclitaxel. To do this we utilised the assemblase scaffold produced in Laboratory Objective #1.
Take a look at Laboratory Objective #3 to see the other pathway we optimised using Assemblase
Introduction
When Paclitaxel was first discovered, significant efforts went into biosynthetically producing Paclitaxel from its natural enzymes. However, due to the complex steps and many enzymes involved, progress has stagnated. Research continues to this day however, as the benefits of biosynthesis are significant. It presents a more sustainable and efficient compared to chemical semi-synthesis that is currently used in industry.
The first stage of our project hence involves improving the rate limiting biosynthesis enzymes, PAM and TycAS563A. PAM catalyses the conversion of ⍺-phenylalanine to 𝛽-phenylalanine2. TycAS563A combines the 𝛽-phenylalanine and CoA thioester, catalysing the production of phenylisoserinyl-CoA3. By co-localising these proteins using the assemblase scaffold we hope to increase the net rate of Paclitaxel formation and in turn improve the overall efficiency of the pathway.
Figure 2. Visualisation of reaction pathway 1. PAM catalyses the conversion of ⍺-phenylalanine to 𝛽-phenylalanine. TycAS563A catalyses the production of phenylisoserinyl-CoA by the addition of the CoA thioester group to 𝛽-phenylalanine.
We plan to express these proteins fused with Spy and Snoop Tags for the attachment to our Assemblase scaffold, thus forming the PAM-SnoopT and TycAS563A-SpyT recombinant protein variants.Gene construction – Design
The following gene construct was designed to enable the cloning and expression of the recombinant proteins PAM-SnoopTag and TycA-SpyTag within a T7 expression system.
Additions to the gene are as follows:
- SnoopTag/SpyTag - The SnoopTag/SpyTag has the ability to conjugate to the SnoopCatcher/SpyCatcher via spontaneous isopeptide bond formation. This allows for the bioconjugation of the protein of interest to any protein fused to a SnoopCatcher.
- Hexahistidine Tag - The histidine residues has a high binding affinity to Ni2+, this enables the purification of the protein of interest using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
- Gibson forward and reverse overhangs - 5’ forward and 3’ reverse gibson overhangs are complementary to that of the pET-19b backbone. This allows for the ligation of the protein of interest into the pET-19b plasmid via Gibson Assembly.
Additionally, GSG linkers are included between the peptide sequences. This flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag system).
Figure 3. Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of PAM-SnoopT and TycA-SpyTag gene constructs. PAM (purple) and TycA (orange) contains a hexahistidine tag (silver) and SnoopTag (yellow) or SpyTag (blue). These are separated by flexible GSG linkers (dark grey). The gene constructs are enclosed within a 5’ gibson forward overhang and 3’ gibson reverse overhangs (red). Image by Linda Chen.
Objectives
This section aims to achieve the following:
- To Clone PAM-SnoopT and TycA-SpyT into pET-19b vectors
- Express and purify PAM-SnoopT and TycA-SpyT fusion proteins
- Obtain enzyme kinetic measurements of enzymes (with and without attachment to the Assemblase scaffold)
Summary of our findings
| Successes | Successfully cloned PAM into pET-19b. |
|---|---|
| Successfully expressed soluble PAM proteins. | |
| Current Tasks | Clone TycA into pET-19b. |
| Perform NMR to test enzyme kinetics of PAM enzyme. | |
| Future Directions | Conjugate PAM and TycA enzymes to ⍺PFD-SpyC and 𝛽PFD-SnoopC scaffold subunits respectively. |
| Obtain enzyme kinetic measurements of both enzymes with and without attachment to Assemblase. |
Results
Cloning
Aim: To assemble PAM and TycA gene constructs (Figure 3) into pET-19b via Gibson Assembly.
Methods:
A typical cloning workflow is summarised in Figure 4. Gene fragments were designed and obtained from Integrated DNA Technologies (IDT). pET-19b plasmid (obtained from Dr Dominic Glover) was linearised using PCR amplification. Ligation of our gene of interest was performed by Gibson Assembly and the ligation products were transformed into competent T7 Express E. coli by heat shock at 42°C for 10 seconds. Transformant colonies were plated onto Ampicillin supplemented Luria Broth Agar plates for selection. Colony PCR was used to screen for colonies containing the recombinant plasmid, PCR products were visualised on a 1% agarose gel. Positive colonies were grown up and plasmid DNA was extracted by miniprep and gene fragment sequenced by Sanger Sequencing.
Figure 4. A typical cloning workflow, describing the vector preparation, insert preparation, ligation, transformation and confirmation steps. Protocol based on instructions from ThermoFisher.
Detailed protocols are available here
Results
The PAM and TycA gene constructs were cloned into pET-19b backbone by Gibson Assembly with a 3X molar excess of insert to vector. The assembly mix was incubated for 1 hour at 37°C before transforming into T7 Express E. coli cells by heatshocking at 42°C. Transformed cells were plated on a Luria Broth (LB) Agar plate supplemented with Ampicillin at working concentration for selection, and incubated overnight at 37°C. Transformant colonies were screened for recombinant plasmids by colony PCR under the following conditions: Initial denaturation at 97°C for 3 minutes, 30X cycles of: denaturation at 97°C for 10 seconds, annealing at 67.6°C for 30 seconds and extension at 72°C for 50 seconds. Final extension at 72°C for 5 minutes. PCR products were run on a 1% agarose gel in 1X TAE Buffer for 1 hour at 100 V. The gel was imaged under transilluminator setting. PCR results revealed the PAM amplicon was approximately 2180 bp in length (Figure 5) and the TycA amplicon was approximately 1000 bp in length (Figure omitted).
Figure 5. Colony PCR gel image of recombinant PAM-pET-19b plasmid. PAM gene was amplified by colony PCR under the following conditions: 97°C for 3 minutes, 30X cycles at: 97°C for 10 seconds, 67.6°C for 30 seconds, 72°C for 50 seconds. Final extension at 72°C for 5 minutes. 10 uL of PCR product was run on a 1% agarose gel at 100 V for 1 hour using 5 uL of 2-log DNA ladder (NEB) as a standard (Lane 1). Single band obtained at approximately 2180 bp.
A single PAM colony was grown up overnight in a 5 mL culture of Luria Broth supplemented with Ampicillin at working concentration. Cells were pelleted and lysed for plasmid purification using QIAGEN miniprep kit. Purified plasmid DNA was sequenced confirmed by Sanger Sequencing, obtaining high sequence homology with the expected PAM gene construct. Sanger Sequencing results revealed some inconsistencies in the bases as summarised in Table 1.
| Base Position | Base change | Quality | Amino acid position | Amino acid change |
|---|---|---|---|---|
| 337 | C-> T | 61 | 106 | R -> C |
| 396 | G -> A | 61 | 123 | P -> P |
| 1107 | A -> - | - | 362 | - |
| 1798 | G -> - | - | 593 | - |
| 2160 | A -> G | 46 | 713 | Stop -> Stop |
Similarly, TycA plasmid DNA was purified and sequencing results revealed low sequence homology to the expected TycA construct, indicating TycA has not been assembled into the pET-19b plasmid.
Discussion
pET-19b vector (provided by Dr Dominic Glover) was linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA Technologies (IDT). The gene constructs were assembled into the pET-19b expression vector at the multiple cloning site via Gibson Assembly with a 3-fold molar excess of insert.
Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C for 10 seconds and plated on Ampicillin supplemented agar plates for selection. This resulted in two (PAM) and zero (TycA) transformed colonies, compared to zero colonies on the linear pET-19b transformed negative control. This was repeated on TycA gene inserts until transformed colonies were detected.
PAM
Two colonies of PAM transformants were screened by colony PCR, where the PAM-2 colony exhibited a single band close to the estimated length of 2180 kb (Figure 5). PAM-2 colony was grown overnight in a 5 mL culture and plasmid DNA was extracted via miniprep. Samples were submitted for sequence confirmation by Sanger Sequencing.
Sequence alignment revealed sequence homology of the PAM-2 colony with our designed gene construct. Thus, PAM has been successfully cloned into pET-19b backbone. However, the gene contains five base inaccuracies. This results in the following amino acid changes: silent mutations at amino acid residue 123 (P) and 713 (Stop), and a missense mutation R106C (Table 1). These detected base changes could be a result of a sequencing error, errors introduced during PCR or errors in the original purchased gene fragment.
To completely understand the effect of the R106C change, further research into the location of this amino acid within the protein by modelling or otherwise could be completed. This would help determine whether the amino acid change would have interfered in the protein folding, or substrate binding of PAM.
TycA
TycA gene inserts were obtained in two fragments (TycA-1 and TycA-2), with a 60 bp overlap between the two fragments. Ligation of both TycA-1 and TycA-2 fragments with pET-19b was attempted via Gibson Assembly. Colony PCR performed on the TycA colonies showed bands which did not align with the theoretical length of 3.383 kb. Sanger Sequencing results confirmed that the colonies submitted did not contain the desired TycA gene insert (Figure omitted).
As TycA is a larger gene fragment (3.3kb), we attempted to increase the incubation time during Gibson Assembly to allow for enough time for the complementary overhangs to properly anneal between the insert and vector.
The following conditions have been attempted: Gibson Assembly incubation – 15 minutes, 60 minutes. The 60 minutes (Figure 6A) incubation produced greater number of colonies compared to the 15 minutes (Figure 6B). This suggests the longer incubation time allows for the ligation of fragments into a functional circularised plasmid. Gibson master mix: commercial compared with home-made . Master mix: commercial had more colonies than the home-made, but not significantly (Figure 6C). Insert to vector: 3X excess, 5X excess.
With multiple repetitions of the Gibson Assembly procedure, the lack of successful recombinant plasmids raises the potential question, that perhaps the gene fragments are not correctly synthesised. We plan to confirm the sequence of our gene fragments empirically, by Sanger Sequencing.
Conclusion
PAM-SnoopTag was successfully cloned into pET-19b and transformed into T7 Express E. coli. Cells are stored in glycerol stocks and ready for expression and purification of the PAM-SnoopTag enzyme.
Attempts to clone TycA have thus far been unsuccessful. In the future, we may attempt to anneal the two gene insert fragments (TycA-1 and TycA-2) by PCR prior to attempting Gibson Assembly. The incubation time during Gibson Assembly could also be increased further to allow enough time for the Assembly to occur between these large gene fragments.
Protein Expression and Purification
Protein expression assay
Cells with pET-19b vector containing PAM insert (as confirmed by colony PCR and sequencing) were grown up in a large-scale overnight culture of Luria Broth containing ampicillin at 37°C with shaking. Once OD600 reached 0.6, protein expression was induced using 0.5 mM IPTG and grown for 18 hours at 15°C. A protein expression assay was performed on a sample of the overnight culture, using a cell-lysis detergent (BugBuster) to separate soluble and insoluble proteins (Figure 7).
Figure 7. Protein expression assay using BugBuster (Novagen) to determine expression of PAM as soluble and insoluble form. A large scale culture of PAM transformants in Luria Broth and ampicillin was induced at OD600 of 0.6 using 0.5 mM IPTG and grown for 18 hours at 15°C.
Purification
Following the confirmation of protein expression using BugBuster gels in Figure 7, attempts were made to purify PAM. The eluted fractions were concentrated 20-fold using an Amicon Ultra-0.5 mL Centrifugal Filters to increase the visibility of any soluble PAM present.
Figure 8. SDS-PAGE of AKTA purification fractions (F3 to F6) for PAM His-tagged protein
Liquid Chromatography with tandem Mass Spectrometry
Soluble protein bands (concentrated fractions 4-6) and total protein lysate bands were excised from the gel of purified fractions seen in Figure 8. These bands were sent for analysis by trypsin digest in conjunction with liquid chromatography- tandem mass spectrometry (LC-MS/MS). This was performed to determine the identity of the protein bands by mapping peptides detected by LC-MS/MS onto the sequence of PAM obtained from sequencing data of the cloned insert.
Figure 9. Peptides detected by LC-MS/MS mapped onto PAM_TAXWC amino acid sequence using MASCOT. A) Total protein lysate sample. B) Soluble protein sample taken from concentrated fractions 4-6 (Figure 8)
A: Total protein lysate sample.
B: Soluble protein sample taken from concentrated fractions 4-6.
Assays for PAM: NMR
Figure 10. Water suppressed 1H NMR spectra of α- and β-phenylalanine. The resonances of the acquired spectra were assigned by referring to the literature and information provided by manufacturers. The change in chemical shift allows the product, β-Phe, to be distinguished from the substrate, α-Phe.
The transfer of the amino group (-NH2) from the α-carbon to the β-carbon changes the hydrogen environments within phenylalanine. Because the proton chemical shifts for α-phenylalanine and β-phenylalanine are different, both the substrate and product can be monitored simultaneously for each NMR spectrum. An internal standard such as 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) can be included to allow for semi-quantitative analysis.
Discussion
PAM
During the large scale grow up of the T7 Express cells containing pET-19b plasmid with a PAM insert, samples were taken to perform an expression assay using BugBuster® Protein Extraction Reagent (Novagen). By performing BugBuster, we were able to determine if our protein of interest was expressed as soluble or insoluble by running samples on an SDS PAGE gel (Figure 7). PAM appeared to be expressed predominately as an insoluble protein. Multiple attempts were made to optimize expression conditions to achieve a higher percentage of soluble protein. Varying IPTG concentration, temperature and expression time were tested however, the solubility of our protein did not appear to improve. Walker et.al (2004) recorded a time of 18 hours at 15°C with 1 mM IPTG1 to be the optimal conditions for PAM expression however, following these conditions we did not yield significant soluble protein. A reason for this could be the His-tag of our protein being on the C-terminus while Walker et.al placed it on the N-terminus1. This may have affected the folding of the protein causing it to be largely insoluble2. Refolding insoluble protein was also explored. Due to the protein having multiple cysteine residues (14), it would have been almost impossible to correctly form all the disulfide bonds of the protein and test optimal refolding buffers and conditions. Another method used to increase the yield of soluble protein was to transform our plasmid into Origami™ 2(DE3)pLysS Competent Cells from Novagen. This is a strain with enhanced ability to form disulfide bonds in E. coli. The trials to achieve this however were unsuccessful, with protein expression assays showing no PAM protein bands despite positive sequencing results.
Following large scale grow up and IPTG induction, cells were lysed by sonication with the soluble fraction purified on a Nickel NTA column via the AKTA Start chromatography system. This produced protein fractions which were concentrated 20-fold using Amicon Ultra-0.5 mL Centrifugal Filters and run on an SDS-PAGE gel (Figure 8). A light band at the same molecular weight as PAM (70kD) was observed and cut out for analysis by LC-MS/MS to determine protein identity. Figure 9 shows the peptides detected by Mass Spectrometry mapped onto the sequence of PAM. This mapping shows a 60% match was achieved using PAM lysate gel sample. This had a large amount of coverage across the sequence indicating PAM is present in the full form. Concentrated purified protein bands showed only a 6% coverage however this is enough to confirm the PAM protein as present in the sample.
Although the protein produced was largely insoluble, a small amount of soluble protein was present as confirmed by mass spectrometry. To obtain more soluble protein, PCR could be used to change the location of the His-tag from the C- to N-terminus of the protein as aforementioned by Walker et.al. 1
Conjugation of PAM-SnoopTag with 𝛽PFD-SnoopCatcher was also tested. This was however unsuccessful, with no conjugation of proteins being present on the SDS-PAGE gel. This is likely due to PAM being in very low abundance. Additionally, high contaminants may hinder the ability for the proteins to conjugate to an appropriate level as to produce a visible conjugation band.
In the future we hope to successfully clone, express and purify TycA as well as, express PAM as a higher concentration of soluble protein. Additionally, we would like to conjugate the enzymes to the Assemblase scaffold proving their functionality using the aforementioned assays.
References
- Walker K, Klettke K, Akiyama T, Croteau R. Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis. Journal of Biological Chemistry. 2004;279(52):53947-53954.