mutagenic oligonucleotide to the template. The mutant strand was synthesized by T4 DNA polymerase prior the
hetero-duplex DNA strands were transformed into the BMH 71-18
mut
S and the cell was grown in a selective
medium. Then, the plasmid was purified from the transformant, and transformed to
in vivo
amplified in
E. coli
JM109.
2. Transformation into
Pichia
cells
The purified recombinant plasmid was linearized with
Sal
I and, then, mixed with the competent
Pichia
cells
prior transferring to a 0.2 cm electroporation cuvette. The mixture was incubated on ice for 5 min and electroporation
was carried out at a voltage of 1500 V, capacitance of 50 µF and resistance of 200
Ω
using an Electroporator II
(Invitrogen). Immediately thereafter, cells were flushed for a couple of times with ice-cold 1M sorbitol, spread on a
minimal dextrose medium (MD) agar plate, and incubated at 30
o
C for 2 days.
3. Screening and expression of recombinant clone
The
Pichia
transformants with phenotype His
+
Mut
+
(hisdine synthesis and methanol utilization plus) were
identified by selective growing on minimal dextrose medium (MD) and minimal methanol medium (MM) agar plates.
Twenty to fifty colonies of His
+
Mut
+
were selected and the recombinants were induced to synthesize TTR as
previously described with 0.5% methanol for 5 days. The culture medium was collected and its supernatant was
analyzed for secreted TTR by SDS-PAGE followed by silver staining.
4. Synthesis in
P. pastoris
of recombinant L110P and V30M
The synthesis was performed using the shake-flask system. A single colony of the recombinant clone was
inoculated in 10 ml of BMGY and the yeast cell was grown up at 30
o
C for 16-18 h. Thereafter, 5 ml of the overnight
culture was transferred into 300 ml of BMGY in a 1-liter flask, and the cells were grown until OD
600
reached 3. The
yeast cells were collected by centrifugation at 2,500 rpm for 5 min at room temperature, re-suspended in 300 ml of
BMMY in a 2-liter flask to a density of 1.0 (at OD
600
). The induction for the synthesis of the recombinant protein was
carried out with 0.5% methanol at 28
o
C, for 7 days. The culture supernatant was collected by centrifugation, and level
of the synthesis of the recombinant TTR was determined by native-PAGE and SDS-PAGE followed by silver staining.
5. Purification of recombinant TTR
The recombinant TTR was purified from the culture supernatant by preparative native-PAGE using Prep
Cell model 491 (Bio-Rad) with discontinuous gel (12% of resolving gel and 4% of stacking gel). The eluting fractions
were collected and TTR in the fractions were determined by native-PAGE followed by silver staining. The fractions
containing TTR were pooled and stored at -20
o
C until used.
6. Molecular weight of TTR subunit
Molecular weight of TTR subunit was determined by SDS-PAGE. The protein was mixed with a solution
containing 2% SDS and 2.5% of
-mercaptoethanol, and incubated at 100
o
C for 30 min prior analysis by SDS-PAGE
was performed, and the protein band was visualized by staining with Coomassie brilliant blue R-250.
6. Amyloid fibril formation
Purified TTR was diluted in 50 mM sodium acetate, pH 4.2, 100 mM KCl and 0.1 mM EDTA to a final
concentration of 0.50
M. The solution, then, was incubated at 37
o
C in dark for 7 days. Thereafter, fibrils were
analyzed by Thioflavin T assay. Fluorescence of the TTR amyloid was measured by spectrofluorometer (Shimadzu
