Biology Reference
In-Depth Information
2.
-subunit cDNA,
extending from the translation initiation codon to an internal
Hind
III site, with
Hind
III and
Sma
I sites flanking the initiation codon, and the 3' end, extending
from an internal
Pst
I site to the translation stop codon, with
Sma
I and
Bam
HI
sites following the stop codon, by PCR. Use these PCR fragments to replace the
5' and 3' ends of the original
Synthesize the 5' end of the human prolyl 4-hydroxylase
α
-subunit cDNA to generate a cDNA without any 5'
and 3' untranslated regions. Ligate the
Sma
I-
Sma
I
α
α
-subunit insert into the
Eco
RI
site of pARG815, generating pARG815
α
.
3.
Replace the signal sequence of the human prolyl 4-hydroxylase
β
-subunit with
the
S. cerevisiae
α
mating factor (
α
MF) pre-pro sequence (
see
Note 9
). Synthe-
size human prolyl 4-hydroxylase
-subunit cDNA, extending from the codon for
the first amino acid after the signal peptide cleavage site to the stop codon and
flanked by
Eco
RI sites, by PCR and ligate into the
Eco
RI site following the
(
β
α
MF) pre-pro sequence of pPIC9, generating pPIC9
β
.
4.
Replace the 3' end of the pro
1(III) cDNA used to generate recombinant
baculovirus pVLrhproCIII (
see
step 2
in
Subheading 3.1.1.
) by a PCR fragment
extending from an internal
Eco
RI site to the translation stop codon, with a
Xba
I
site following the stop codon. Ligate the
Bgl
II-
Xba
I pro
α
α
1(III) cDNA into the
Eco
RI-
Xba
I site of pPICZ B, generating pPICZ Bpro
α
1(III).
3.2.2. Generation of the Recombinant Pichia Strain
and Expression of the Recombinant Human Type III Procollagen
1.
To obtain a recombinant
P. pastoris
strain expressing human prolyl 4-hydroxy-
lase, linearize pARG815
with
Dra
III and
Stu
I, respectively, and
cotransform into the
his4, arg4 P. pastoris
host strain by the electroporation
method
(15)
. Culture 5 mL of the
his4, arg4 P. pastoris
host strain in YPD in
a 50-mL conical tube at 30
α
and pPIC9
β
C overnight. Inoculate 500 mL of YPD with
0.1-0.5 mL of the overnight culture and grow to an OD
600
of 1.3-1.5 (
see
Note 10
).
Centrifuge the cells at 4
°
C at 1 500
g
for 5 min and wash twice, with 500 mL and
250 mL of ice-cold sterile water. Resuspend in 20 mL of ice-cold 1
M
sorbitol,
centrifuge, and resuspend in ice-cold 1
M
sorbitol to obtain a final volume of 1.5 mL.
Mix 3
°
L of
the
P. pastoris
cells, transfer to an ice-cold electroporation cuvet and incubate on
ice for 5 min. Pulse the cells using the parameters 1500 kV, 25
µ
L (approx 0.6
µ
g) of the linearized pARG815
α
and pPIC9
β
with 40
µ
Ω
(
see
Note 11
), and add 1 mL of ice-cold 1
M
sorbitol immediately afterwards.
Spread aliquots of 50-200
µ
F, and 400
C
until colonies appear. Repeat selection for His
+
, Arg
+
colonies twice by streaking
single colonies on MD plates.
µ
L of the cells on MD plates and incubate at 30
°
Culture the recombinant His
+
, Arg
+
colonies obtained above at 30
2.
C in 25 mL of
BMGY in 250 mL shaker flasks to an OD
600
of 5-10. Centrifuge the cells at 1
500
g
for 5 min and resuspend to an OD
600
of 1-3 in BMM to induce expression.
Add methanol every 24 h to a final concentration of 0.5%. Harvest cells after a
60-h methanol induction, wash once and resuspend to an OD
600
of 250-350 in
cold 5% glycerol in 50 m
M
sodium phosphate buffer, pH 7.4. Add an equal
°
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