Linearisation of constructs was necessary prior to transformation into Pichia pastoris cells to stimulate homologous recombination with the host genome as discussed in section 3.1.3.2. This reaction was carried out in a mixture consisting of ~15pg of construct DNA, 20 units of Prne\, 1 Ox restriction enzyme buffer and Milli Q water in a total reaction volume of lOpl. The mixture was incubated at 37°C for one hour then cleaned using the Wizard DNA Clean-up kit as described in section 2.2.9.
3.4.3.3 Transformation of Pichia pastoris with linearised construct
The linearised construct DNA was used to transform competent Pichia pastoris cells (GSl 15, KM71 and X-33) using the method o f electroporation. All reagents used for the electroporation procedure were kept cold on ice. Approximately 7p,g o f construct DNA were mixed with 100p,l of competent P/c/izTz pastoris cells in a 1.5ml eppendorf tube before transferring the mixture to a 2mm electroporation cuvette. The mixture was electroporated using a charging voltage of 1500V, capacitance of 25p,F and resistance o f 200Q. 1ml of ice cold 1M sorbitol was added immediately to the electroporated cells and the mixture plated out onto YPDS agar plates with 100p,g/ml zeocin. Plates were incubated at 30°C for up to three days or until colonies had formed. Glycerol stocks o f the control P/c/î/a pastoris
strains, GSl 15/Muf Albumin (Muf) and GSl 15/pPICZ/lacZ^ut^ (Mut^), were also streaked onto YPDS agar plates containing zeocin. The M uf control was a negative control for growth on zeocin medium while the Mut^ was a positive control for growth on this medium.
3.4.3.4 Determination of the methanol phenotype of transformant colonies
This was carried out to ensure that GSl 15 and X-33 transformants were still Mut^ phenotype when grown on methanol-containing medium. Zeocin resistant colonies for all
three transformant strains of Pichia pastoris (GSl 15, KM71 and X-33), with the insert in
the correct orientation, were selected for methanol phenotype screening. Colonies were picked with a sterile loop and resuspended in 200|il of IM sorbital in a 96-well microtitre tray. A template grid was used to draw a grid on the MDH and MMH agar plates (Figure 3.4). Resuspended colonies were then streaked in a regular pattern onto a dedicated grid ensuring MMH plate was streaked before the MDH plate. A sterile toothpick was used for each colony. Control strains for both Mut^ and M uf were also streaked on both plates. These were the GS 115/M uf Albumin and GS 115/pPICZ/lacZ/Mut^ control strains. The plates were incubated at 30°C for up to four days but were examined for growth at day 2 and day 4.
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H Q Q Q Q Q Q H T
Figure 3.4 Grid template used for streaking Pichia pastoris transformant strains. T h i s t e m p l a t e w a s u s e d to m a r k b o t h t h e M D H a n d M M H p l a t e s f o r m e t h a n o l p h e n o t y p e d e t e r m i n a t i o n .
3.4.4 Expression of the G S llS /M u f Albumin (M uf) and transform ant Pichia
pastoris cells
3.4.4.1 Secreted expression of albumin
A single colony for the GS 115/Mut" Albumin (Muf) was innoculated into 25ml of BMGY media and incubated at 30°C shaking incubator until growth of the yeast gave an absorbance of between 2 - 6 units when measured at 600nm using a UV/Vis spectrophotometer. The cells would be in log phase of growth at this stage. Once the cells
had reached the required they were centrifuged at 2000gat room temperature for five
minutes. The supernatant was discarded and the cell pellet resuspended in BMMY to an OD(,oo of 1.0. The flasks were returned to the 30°C shaking incubator to allow expression to occur in the presence of the methanol. These cultures were allowed to grow for up to 5 days and 100% methanol (0.5%v/v) was added every 24 hours to maintain induction. 1 ml samples of the cultures were taken at the time points: 0, 24, 96 and 120 hours after induction, i.e. switching of media from BMGY to BMMY. These were centrifuged at maximum speed in a bench top centrifuge and the supernatant separated from the cell pellet. Both were retained and stored at -70°C for analysis by SDS PAGE.
3.4.4.2 Expression studies on Pichia pastoris transformant colonies
GS115/KM71/X-33-PDE1A1 transformant colonies, as well as cells not containing the insert, were treated in the same way as the expression of the GSl 15/Mut®-Albumin for the secreted expression of recombinant PDEIAI. Expression was carried out for up to 5 days and 1ml samples of the culture media removed every 24 hours for monitoring protein expression. These were centrifuged as before and both the cleared media and cell pellet samples retained for analysis; both were stored at -70°C until needed. The cells were lysed using breaking buffer containing PMSF and glass beads (425-600 microns). This mixture was vortex mixed for 4 - 5 times to break up the yeast cells and then centrifuged at maximum speed (14,000 rpm) in a benchtop centrifuge. Cleared total cell lysate (supernatant) was separated from the cell debris and retained for analysis by PDE activity assays as described in section 2.2.11. Western blot analysis using CaM-PDE antibody was carried out on both the cleared culture media as well as the cleared total cell lysate samples as described in section 2.2.14.
3.5 Results