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DBAT^G38R/F301V-SnoopT-His

Introduction

DBAT-Snooptag-His consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) fused to a short C-terminal polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of DBAT which was used, originated from Taxus cuspidata (Japanese yew), with a double mutation of G38R/F301V (2). The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.

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

This protein naturally participates in the synthesis of baccatin III from 10-deacetyl-2-debenzoylbaccatin III, where it catalyses the final acetylation. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)

Figure 2:

Characterisation

pET19b vector was provided by Dr Dominic Glover and 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 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 nine (DBAT) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of DBAT were screened by colony PCR, where DBAT-1 obtained a single band of estimated molecular weight ______ (approx. 1.4kb)[MT1] (Figure 3).

Figure 3:

DBAT-1 colony was grown overnight in a 5mL culture and plasmid DNA was extracted by miniprep. Samples were submitted for sequence confirmation by Sanger sequencing, obtaining the sequence utilising forward and reverse primers. Sequence alignment revealed 100% sequence homology of the DBAT-1 colony. The DBAT-SnoopT gene fragment was aligned from base 23 to 1195 (in the D1-F sequence alignment file) and ____ to _____ [MT3] (in the D1-R sequence alignment file). This confirms that DBAT-SnoopT has been successfully assembled and transformed within DBAT-1 colony and was stored in glycerol stocks and prepared for protein expression and purification.

Protein Expression and Purification

Protein expression assay

Cells containing a plasmid with the DBAT insert were grown up with a sample of this being used to perform a protein expression assay. Additionally, Bug buster was used to separate soluble and insoluble proteins.

Figure 4: Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.

Purification

Following the confirmation of protein expression using bug buster gels, attempts were made to purify DBAT.

Figure 5: SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein

Liquid Chromatography with tandem mass spectrometry

Soluble protein bands (fractions 4-7) as well as, a total protein lysate band at the same predicted molecular weight as DBAT were excised from the Figure 5 gel and sent for analysis. Trypsin digest followed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) confirmed the identity of the protein bands as DBAT, the peptides of which are shown in figure 6.

Figure 6: The Liquid Chromatography with Tandem Mass Spectrometry analysis of suspected DBAT protein bands excised form Figure ? protein gel. A: Total protein lysate sample. B: Soluble protein sample taken from fractions 4-7.

Validation

Conjugation

Ellman’s Reagent Assay

A standard curve measuring absorbance vs differing concentrations of CoA-SH was constructed (Figure 7) with a computer generated linear trendline.

Figure 7: Standard curve of TNB absorbance measured at differing CoA-SH concentrations. Included is the computer generated linear trendline with Cartesian equation and R value.

A discontinuous assay was conducted in triplicate at 15-minute interval time points for a total of 90 minutes. Mean sample absorbance – Mean blank absorbance was calculated for each time point. Excel was then used to create a graph containing a computer-generated linear trendline (figure 8)

Figure 8: Endpoint assay of change in TNB absorbance over 90 minutes, measured every 15 minutes. Included is the computer generated linear trendline with Cartesian equation and R value

The amount of TNB produced in 1 minute was calculated by c= ∆A/(εl) where c is the concentration change of TNB (M), ∆A is the change in absorbance in 1 minute, molar extinction coefficient ε= 14 150 M-1cm-1, and pathlength l= 1 cm. (2) The result was then divided by 60 to get the change in TNB concentration per second. (3)

The number of moles of TNB produced per second was calculated by n= cV where c is the concentration change calculated from the step above, and V = 350 x 10-6 L.

The rate of catalysis was calculated by (moles of TNB produced per second)/(moles of enzyme) as TNB is formed in a 1:1 molar ratio with CoA-SH produced. This gave us an estimated Kcat of 0.0056 (1/s)