Part:BBa_K3075000
PAM-SnoopT-His
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 498
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 498
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 498
Illegal BamHI site found at 2131 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 498
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 498
Illegal NgoMIV site found at 393 - 1000COMPATIBLE WITH RFC[1000]
Introduction
PAM-Snooptag-His consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from Taxus wallichiana var. chinensis was utilised. (1)
Figure 1:
The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.
Usage and Biology
Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.
Figure 2:
Characterisation
pET19b vector was provided by Dr Dominic Glover and was linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b 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 42C and plated on Ampicillin supplemented agar plates for selection. This resulted in two (PAM) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control.
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 2.18 kb (Figure 3). 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.
Figure 3: Colony PCR gel image of recombinant PAM-pET19b plasmid. PAM gene was amplified by colony PCR at Tm = 67.6°C and extension time 50 seconds, else as per protocol <link>. 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 at ~2.2 kb.
Sequence alignment reveal sequence homology of the PAM-2 colony with our designed gene construct. Thus, PAM has been successfully cloned into pET19b 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.
Table 1: Base inconsistencies between Sanger sequencing results of PAM-2 and the desired PAM-Snooptag-His sequence.
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.
Protein Expression and Purification
Protein Expression assay
Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up with a sample of this used to perform a protein expression assay. Bug buster was then used to separate the soluble and insoluble proteins.
Figure 4: Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.
Purification
Following the confirmation of protein expression using bug buster gels in Figure 4, 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 5: 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 5. These bands were the same predicted molecular weight as PAM. The bands were sent for analysis by Liquid Chromatography with 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 6: LCMSMS analysis of suspected PAM protein bands excised form Figure 8 protein gel. A: Total protein lysate sample. B: Soluble protein sample taken from concentrated fractions 4-6.
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