The purity and concentration of the PCR product was assessed via electrophoresis using a 1% agarose gel. 1% (w/v) agarose powder was added to 100 ml of 1X TAE buffer (0.484% (w/v) Tris-base, 1.142% (v/v) glacial acetic acid, and 0.029224% (w/v) EDTA) and dissolved by heating in a microwave oven for 3 minutes with periodic agitation every 30 seconds in a 250 ml glass flask. When the agarose gel solution was cooled to 50 °C, 10 µl of SYBR® Safe DNA gel stain (Invitrogen) was added to the agarose gel solution and the mixture poured into a gel caster. 5 µl of the PCR products
48 were loaded into each well and run at 25 V until the dye front has migrated the length of the gel. Agarose gels were visualised using a UV Transilluminator (Syngene). The PCR products were purified using a QIAquick Gel Extraction kit (Qiagen) to remove the excess dNTPs and the original pGEM vector. Quantification of purified DNA samples was conducted sing a NanoDrop 3300 (Thermo Scientific).
The PCR product was then prepared as per manufacturers protocols for the respective Xa/LIC kit to generate LIC compatible overhangs.
2.2.6 Plasmids
2.2.6.1 Expression in Escherichia coli
The pET vector series (Merck) for cloning was extensively used throughout the cloning process. The two major vectors used were the pET-32 Xa/LIC vector and the pET-30 Xa/LIC vector (Fig 2.1). The pET-32 Xa/LIC vector fuses the target protein with a 109aa thioredoxin (trx.tag) fusion partner. The vector also contains a His-tag and an S- tag to facilitate purification. The thioredoxin fusion partner has been proven to enhance the proper formation of disulphide bonds in cysteines-rich proteins. The vector also contains a Factor Xa cleavage site (ile-glu/asp-gly-arg) for the easy removal of the fusion partner and His-tag from the target protein.
The pET-30 Xa/LIC vector contains a His-tag and an S-tag without the thioredoxin fusion partner. It is primarily used for the expression of the target protein when the action of thioredoxin would be undesirable. The pET-30 Xa/LIC vector also has a Factor Xa Cleavage site which allows for the removal of the His-tag and S-tags.
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Step Pfu KOD
Temperature (°C) Time No. of Cycles Temperature (°C) Time No. of Cycles
Initial Denaturation 94 2 min 1 94 2 min 1
Denaturation 94 1 min 30 94 15 sec 30
Annealing 55 30 sec 60 30 sec
Extension 72 2 min 72 1 min
Final Extension 72 5 min 1 72 5 min 1
Soak 4 indefinite 1 4 indefinite 1
Table 2.1: Temperature cycling conditions for PCR. PCR reactions were conducted in 50µl volumes using either Pfu or KOD polymerase. Initial annealing temperature was
50 Figure 2.1: Cloning/expression region of pET-30 Xa/LIC and pET-32 Xa/LIC (Novagen). The
plasmids pET-30 Xa/LIC and pET-32 Xa/LIC were used to reduce process error due to ligation. pET-32 Xa/LIC allows for the production of a thioredoxin fusion protein with a Factor Xa cleavage site that allows for removal of the thioredoxin tag. Thioredoxin aids in the proper folding of cysteine-rich proteins. Use of the lac operator in both plasmids allow for highly-regulated inducible expression using IPTG in E. coli. Figure adapted from Novagen manual for pET-30 Xa/LIC and pET-32 Xa/LIC kits.
51 2.2.6.2 Expression in Pichia pastoris
For expression in P. pastoris, the pPICZα A vector (Invitrogen) and the pPIC9k vector (Invitrogen) were used (Fig 2.2).
The pPICZα A vector contains the S. cerevisiae α-factor secretion signa (Fig 2.3). This allows for the secretion of folded proteins from P. pastoris. The vector also contains a C-terminal myc epitope tag that allows for detection of low levels of protein expression through western blot. The presence of a C-terminal polyhistidine tag also allows for purification of the target protein through metal affinity chromatography. SacI and PmeI restriction sites are also present at the aox1 locus to allow for linearization of the plasmid and integration by homologous integration. The pPICZα A vector also contains the Zeocin™ resistance (Sh ble) gene which can be used to select for transformants.
The pPIC9k vector also contains the S. cerevisiae α-factor secretion signal (Fig 2.4). The presence of a C-terminal polyhistidine tag also allows for purification of the target protein through metal affinity chromatography. SacI, SalI and BglII restriction sites are also present at the aox1 locus to allow for linearization of the plasmid and integration by homologous integration. The pPIC9k vector contains the kanamycin resistance gene, which results in low expression levels and hence results in the need for multicopy integrants. Resistance to Geneticin® (G418) conferred on the strain is dose-dependent and allows for selection of multicopy integrants based on the concentration that strains are resistant towards.
Choice of SacI, SalI and BglII restriction sites allows for directed integration of the gene into specific loci (i.e. within the AOX1 or HIS4 genes) in the yeast genome to generate different phenotypes in the yeast cell strains.
52 Figure 2.2: cloning/expression region of pPICZα and pPIC9k. Use of both the pPICZα and the
pPIC9k plasmid allow for pAOX1 regulated expression of heterologous protein. The pPICZα plasmid confers Zeocin™ resistance to successful transformants via the sh ble gene whereas the pPIC9k plasmid confers geneticin® (G418) resistance to successful transformants via the kanR gene. The addition of the
α-factor signal sequence allows for secreted expression and the Kex2 cleavage site allows for proper post-production processing of the signal sequence in both plasmids. pPICZα also encodes a c-myc epitope and a his-tag to allow for verification and purification through western blot and IMAC. Plasmid diagrams adapted from plasmid manuals (Invitrogen)
53 Figure 2.3: Multiple Cloning Site of pPICZα A: The pPICZα A plasmid allows for soluble expression
in P. pastoris. Constructs should be designed so that it is in frame and downstream of the α-factor signal sequence as well as with the C-terminal peptide. Alternative plasmids pPICZα B and C are available with +1 and +2 frame shifts to allow for flexibility in cloning. AT-rich regions can cause premature termination, as such, if AT-rich regions are present in the gene-of interest, changing the sequence to make it more amenable for expression in P. pastoris might be required for successful expression (Scorer, Buckholz et al. 1993). Plasmid diagrams adapted from plasmid manuals (Invitrogen)
54 Figure 2.4: Multiple Cloning Site of pPIC9k: The pPIC9k plasmid allows for soluble expression in P. pastoris. Constructs should be designed so that it is in frame and downstream of the α-factor signal sequence. AT-rich regions can cause premature termination, as such, if AT-rich regions are present in the gene-of interest, changing the sequence to make it more amenable for expression in P. pastoris might be required for successful expression (Scorer, Buckholz et al. 1993). Plasmid diagrams adapted from plasmid manuals (Invitrogen)
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