Biology Reference
In-Depth Information
volume of glass beads and vortex the samples 8 times for 30 s at 4
°
C with 30 s
intervals. Centrifuge at 15,000
g
for 30 min at 4
C, collect the supernatants and
screen for expression of the prolyl 4-hydroxylase
°
-subunits and for the
amount of prolyl 4-hydroxylase activity as in
step 2
of
Subheading 3.1.3.
Select a
recombinant prolyl 4-hydroxylase expressing strain and term it
α
- and
β
α
/
βα
-MF.
3.
Generate a recombinant strain coexpressing human prolyl 4-hydroxylase and
pro
α
1(III) chains by transforming
Pme
I-linearized pPICZ Bpro
α
1(III) into the
-MF strain by electroporation as in
step 1
. Select the transformants in YPD
(+ 100
α
/
βα
g/mL zeocin) (
see
Note 12
). Culture the cells, induce and harvest as in
step 2
, and assay expression of human prolyl 4-hydroxylase and type III
procollagen in the soluble fraction of cell lysates as in
step 2
of
Subheading
3.1.3.
(
see
Note 13
). Select a recombinnt human type III procollagen producing
strain based on these assays and term it
µ
α
/
βα
-MF/pro
α
1(III).
4.
For the production of recombinant human type III procollagen, culture the
α
/
βα
-
MF/pro
1(III)
Pichia
strain in shaker flasks and induce with methanol. Harvest
cells 60 h after methanol induction and brake with glass beads as in
step 2
. The
purified recombinant type III collagen produced in shaker flasks in our experi-
ments was essentially identical in its amino acid composition to nonrecombinant
human type III collagen, except that the degree of 4-hydroxylation of the proline
residues was 44.2% whereas the corresponding value for nonrecombinant
type III collagen is 51.6%
(8)
. However, fully hydroxylated recombinant human
type III procollagen can be produced in the
α
1(III)
Pichia
strain in
bioreactors under optimal oxygenation conditions (unpublished results). The
Pichia
-derived recombinant human type III collagen contains no hydroxylysine,
whereas nonrecombinant type III collagen has five hydroxylysine residues per
1000 amino acids
(8)
.
α
/
βα
-MF/pro
α
4. Notes
1.
In the p2Bac and pVL1392 vectors, the ATG translation initiation codons of
polyhedrin and p10 have been altered to ATT, which means that the inserts must
provide their own ATG initiation codons. The 5' cloning site in the inserts should
be as close to the translation initiation codon as possible (less than 100 bp)
(20)
.
Because translation starts from the first ATG codon downstream of the polyhedrin
and p10 promoters, no additional ATG codons should exist upstream of the ini-
tiation codon of the gene of interest. For example, the polyhedrin multicloning
site in the p2Bac vector has a
Nco
I recognition sequence (CCATGG), and, there-
fore, none of the downstream cloning sites can be used. The length or sequence
of the 3' untranslated region of an insert usually has no effect on the expression
levels. The p2Bac and pVL1392 vectors provide the polyhedrin or SV40
polyadenylation signals, and therefore the genes of interest do not have to include
any polyadenylation signals.
2.
BaculoGold DNA is a modified baculovirus DNA which contains a lethal dele-
tion
(20)
, and viable virus particles are obtained only by cotransfection with a
complementing baculovirus expression vector. When BaculoGold DNA is used,
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