Anillo Oriente

The PDEIAI ORF was subcloned into pPICZaC as detailed in section 3.4.2 and the

product used to transform cells as described in section 3.4.3. Transformed

cells were grown on YPDS agar containing zeocin.

3.5.1.1 Screening transformant colonies

Pichia pastoris transformant colonies were selected on YPDS agar containing zeocin.

Twelve single colonies were selected for DNA purification followed by enzyme restriction

analysis using HindWi. Digestion with this single enzyme was expected to give two DNA

fragments of sizes 0.9 and 4.3 kb for the PDEIAI insert cloned into pPICZaC in the correct orientation (Table 3.2). Where the insert was cloned in the reverse orientation, 1.3 and 3.9 kb fragments were expected following digestion w i t h ( T a b l e 3.2). Figure 3.5 shows

constructs with the insert in the reverse orientation (indicated by the black stars in Figure 3.5) and one construct with the insert in the correct orientation (indicated by the solid red star in Figure 3.5). Figure 3.6.1 shows the enzyme restriction analysis carried out on the

single construct with the insert in the correct orientation using a combination of Hindill,

BamYU. and EcoBl restriction enzymes. This was also carried out for a construct with the

insert in the reverse orientation for comparison. Table 3.2 summarises the DNA fragments expected following enzyme restriction analysis of the construct with the insert in the correct and reverse orientation. A schematic showing the constmcts with the insert in the correct and reverse orientation together with the DNA fragments sizes following restriction enzyme digestion has already been described in Figures 3.2.1 and 3.2.2.

Table 3.2 DNA fragment sizes for pPICZaC-PDElAl constructs with the insert in the

correct (PDEI) and reverse (PDEIR) orientation.

C o n s t r u c t R e s t r i c t i o n e n z y m e (s) D N A f r a g m e n t s i z e s ( k b )

P D EI (correct orientation) BamWl + HindWl 0 . 9 + 1.5 + 2.8

HindlU 0.9 + 4.3

E coR I+ H indlll 0.3 + 0 . 6 + 4.3

P D E I R (reverse orientation) Bam H l + NindlU 1.3 + 1.1 +2. 8

H indlll 1.3+ 3.9

☆ ☆ ☆ ☆ ★ 2 3 4 5 6 7 8 9 10 11 12 13

< - 1 . 3 k b < - 0 . 9 k b

Figure 3.5 Screening transformant colonies for the presence of the construct (s)

containing the PDEIAI insert using HindiW enzyme restriction analysis. D i g e s t i o n s a m p l e s w e r e a n a l y s e d o n 1 % a g a r o s e g e l s . L a n e 1 = 1 k b D N A M a r k e r l a n e w i t h s i z e s i n d i c a t e d o n t h e le f t ; L a n e s 5, 7, 8, 10 a n d 13 = c o n s t r u c t D N A w i t h t h e i n s e r t in t h e r e v e r s e o r i e n t a t i o n ( P D E I R ; i n d i c a t e d b y t h e b l a c k s t a r s ) , f r a g m e n t s i z e s i n d i c a t e d b y t h e o p e n b l a c k a r r o w s ; L a n e 12 = c o n s t r u c t D N A w i t h P D E I A I i n s e r t in t h e c o r r e c t o r i e n t a t i o n ( P D E 1 ; i n d i c a t e d b y t h e s o l i d r e d s t a r ) , f r a g m e n t s i z e s i n d i c a t e d b y t h e o p e n r e d a r r o w s .

1 2 3 4 5 6 7 8

PDE1 PD E IR PDEI P D E IR PDE1 PDE1R

Figure 3.6.1 Confirmation of the PDEIAI orientation in the construct pPlCZaC- PDElAl using enzyme restriction analysis. S a m p l e s w e r e a n a l y s e d o n i % a g a r o s e g e l s f o l l o w i n g r e s t r i c t i o n a n a l y s i s w i t h a c o m b i n a t i o n o f H i n d \ \ \ , B a m \ \ \ a n d E ' c o R l r e s t r i c t i o n e n z y m e s . L a n e s 1 a n d 8 = 1 k b D N A M a r k e r s w i t h s i z e s i n d i c a t e d o n t h e le f t ; L a n e s 2 a n d 3 = P D E I a n d P D E I R r e s p e c t i v e l y f o l l o w i n g d i g e s t i o n w i t h B a m W \ a n d H in d W V , L a n e s 4 a n d 5 = P D E I a n d P D E 1 R r e s p e c t i v e l y f o l l o w i n g d i g e s t i o n w i t h H in d W V , L a n e s 6 a n d 7 = P D E I a n d P D E I R r e s p e c t i v e l y f o l l o w i n g d i g e s t i o n w i t h £ c o R I a n d

3.5.1.2 Automated DNA sequence analysis

DNA sequence analysis was carried out on the construct with the insert in correct orientation using the reverse primer LI 1 as described in section 3.4.2.4. The sequence analysed included the sequence at the N-terminal portion of the ORF and encompasses the region between the two calmodulin-binding domains, the first calmodulin-binding domain and the cloning junction between the N-terminal ORF and the vector. Figure 3.6.2 shows a portion of the sequence at the cloning junction between the N-terminal ORF and the vector.

O R F

I

:T . T CT CT C G ■ G G G „ G G G C T G . G : T C G T G - . T T C , C| . I G G A T G .. C G : T G ! L L .. 1 L , G G , A G , A L I'

Figure 3.6.2 DNA sequence analysis of pPlCZaC-PDE 1 Al p d ei a i i n s e r t is s h o w n in t h e o p e n b o x w i t h t h e r e d a r r o w m a r k i n g t h e p o s i t i o n o f t h e O R F .

The sequence analysed showed no deviations from the sequence obtained by Clapham and Wilderspin (2001).

3.5.2 Methanol phenotype determination for the transform ant Pichia pastoris

strains

The methanol phenotype of the transformant strains {P. pastoris-?DE 1 ) was carried out to ensure that the AOXl gene was not disrupted following integration of the expression cassette. There were two GSl 15, twenty X-33 and one KM71 transformant colonies which were screened for methanol phenotype. Both GSl 15 and X-33 strains have an intact^OA/ gene so are able to grow on methanol-containing media (MMH) with a generation time of approximately 5 hours. Transformation of the KM71 strain of P. pastoris with construct DNA produced only one transformant colony so this was also screened for methanol phenotype. This strain was expected to show a slower growth on MMH media since it has

a disrupted AOXl gene and has a generation time of approximately 30 hours. Table 3.3 summarises the results for the methanol phenotype screening. Growth is denoted as “+” symbols and represent an estimation of the cell growth at days 2 and 4. All three transformant strains showed growth on dextrose media (MDH). GSl 15 and X-33 transformant strains showed a faster growth than the KM71 transformant strain on methanol

media (MMH) as expected since the KM71 strain has a disrupted gene as discussed

earlier (3.1.3.1 and 3.1.3.3). The faster growth rate of the GSl 15 and X-33 transformant

colonies confirmed that the AOXl gene was intact. Both GSl 15 transformant colonies,

twenty X-33 transformant colonies as well as the single KM71 transformant colony were selected for expression studies. Strains of P. pastoris not containing construct DNA were used as controls for expression of background proteins, including any endogenous PDE activity.

Table 3.3 Methanol phenotype determination of transformant P. pastoris strains.

T r a n s f o r m a n t s t r a in G r i d / c o l o n y n u m b e r M D H M M H D a y 2 D a y 4 D a y 2 D a y 4 X-33-PDE1 1 - 2 0 - KM71-PDE1 21 - GS115-PDE1 2 2 - 2 3 -

3.5.3 Secreted expression of G Sl 15/M uf-Albumin (M uf)

The strain GS 115/Mut^ contained an expression cassette comprising of the gene for albumin, in frame with the a-factor signal sequence, together with the zeocin resistance gene. Expression was carried out using this strain to establish the Pichia pastoris

expression system for the expression of secreted proteins. 1ml samples of the culture were removed at the time points 0, 24, 96 and 120 hours, following the switch of media from BMGY to the expression media, BMMY. These samples were centrifuged to remove the cells and the cleared media analysed on 20% SDS PAGE. Figure 3.7 shows the results for these samples.

KDa

M ^ 1