Yeast

Yeast 2001; 18: 797806.

DOI: 10.1002/yea.730

Research Article

High-level production of human type I collagen in the

yeast Pichia pastoris
Minna Nokelainen1, Hongmin Tu1, Annamari Vuorela1, Holger Notbohm2, Kari I. Kivirikko1 and
Johanna Myllyharju1*
1
2

Collagen Research Unit, Biocenter and Department of Medical Biochemistry, PO Box 5000, FIN-90014 University of Oulu, Finland
Institute for Medical Molecular Biology, Medical University of Lubeck, D-23538 Lubeck, Germany

* Correspondence to:
J. Myllyharju, Department of
Medical Biochemistry, University
of Oulu, PO Box 5000,
FIN-90014 University of Oulu,
Finland.
E-mail: johanna.myllyharju@oulu.

Received: 31 October 2000

Accepted: 31 January 2001

Abstract
Four human genes, two of them encoding the proa1 and proa2 chains of type I procollagen
and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated
into the genome of Pichia pastoris. The proa1 and proa2 chains expressed formed type I
procollagen molecules with the correct 2 : 1 chain ratio, and the 4-PH subunits formed an
active enzyme tetramer that fully hydroxylated the proa chains. Chains lacking their N but
not C propeptides formed pCcollagen molecules with the 2 : 1 chain ratio and, surprisingly,
the expression levels of pCcollagen were 1.53-fold relative to those of procollagen. Both
types of molecule could be converted by pepsin treatment to collagen molecules that
formed native-type brils in vitro. The expression levels obtained for the pCcollagen using
only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l,
indicating that it should be possible to optimize this system for high-level production of
recombinant human type I collagen for numerous medical applications. Copyright # 2001
John Wiley & Sons, Ltd.
Keywords:

Pichia pastoris; collagen; procollagen; prolyl 4-hydroxylase

Introduction
The collagens are a family of extracellular matrix
proteins that play a dominant role in maintaining
the structural integrity of various tissues. The
molecule of type I collagen, the most abundant
collagen, is a long, thin rod. It consists of two
identical a1(I) chains and one a2(I) chain that are
coiled around one another in a triple-helical
conformation. The molecule is rst synthesized in
the form of a larger precursor that needs to be
processed by cleavage of the N and C propeptides
present in its proa1(I) and proa2(I) chains. The
collagen molecules then self-assemble into crossstriated brils in which each molecule is displaced
by about one-quarter of its length relative to its
nearest neighbour along the axis of the bril (for
reviews on collagens, see Kielty et al., 1993;
Prockop and Kivirikko, 1995; Kadler et al., 1996).
In addition to the type I collagen heterotrimer,
many tissues contain small amounts of an [a1(I)]3
homotrimer (Kielty et al., 1993). Early renaturation
Copyright # 2001 John Wiley & Sons, Ltd.

experiments (Tkocz and Kuhn, 1969) and recent

recombinant expression studies in insect cells
(Myllyharju et al., 1997) have demonstrated that
the type I proa and a chains are able to form both
[a1(I)]2a2(I) heterotrimers and [a1(I)]3 homotrimers,
although the former are favoured, but they form no
[a2(I)]3 homotrimers (Myllyharju et al., 1997; Lees
et al., 1997).
Type I collagen is now used in numerous medical
applications as a biomaterial and a delivery system
for many drugs (Ramshaw et al., 1996; Pachence,
1996; Ruszczak and Schwartz, 1999). In addition,
all gelatins are made from type I collagen. The
collagens used in all these applications have been
isolated from animal tissues and are therefore liable
to cause immunologic reactions in many human
subjects and also carry a risk of disease-causing
contaminants. It is obvious, therefore, that an
efcient large-scale recombinant system for the
production of human type I collagen would have
numerous applications.
Most systems that are now used for the

M. Nokelainen et al.

798

large-scale production of recombinant proteins

cannot be applied as such to the production of
human type I collagen, as bacteria and yeasts
(Vuorela et al., 1997) have no prolyl 4-hydroxylase
(4-PH) activity, and as insect cells (Lamberg et al.,
1996) and the mammary gland (John et al., 1999)
have insufcient amounts of it. This enzyme plays
a crucial role in the synthesis of all collagens, as
4-hydroxyproline residues are essential for the
formation of molecules with stable triple helices.
Therefore, the recombinant collagen polypeptide
chains produced in most systems will remain in the
form of a non-triple-helical, non-functional protein.
The vertebrate 4-PHs are a2b2 tetramers in which
the b subunit is identical to the enzyme and
chaperone protein disulphide isomerase (PDI)
(Kivirikko and Myllyharju, 1998; Kivirikko and
Pihlajaniemi, 1998).
We have recently demonstrated that coexpression of the proa1(III) chains of human type
III procollagen with the two types of subunit of
human 4-PH in the yeast Pichia pastoris leads to the
formation of properly folded homotrimeric type III
procollagen molecules (Vuorela et al., 1997). The
level of hydroxylation of proline residues obtained
in shaker ask cultures ranged up to about 85% of
that found in the non-recombinant type III collagen, and the expression levels ranged up to 15 mg/l
(Vuorela et al., 1997). The triple-helical procollagen
molecules predominantly accumulated within the
endoplasmic reticulum of the yeast cell (KeizerGunnink et al., 2000) and were not secreted into the
medium (Vuorela et al., 1997). The same coexpression principle has also been used for the
production of an engineered, shortened form of a
type I proa chain in mouse milk at levels of up to
50200 mg/l (John et al., 1999).
We investigated here whether it is possible to
adopt the 4-PH co-expression strategy for high-level
production of heterotrimeric and fully hydroxylated
human [a1(I)]2a2(I) type I collagen molecules in
P. pastoris. The development of such a system
would require co-expression of four human DNAs
in each yeast cell. In addition, we studied whether
the use of a fermenter equipped with an O2 supply
system would eliminate the deciency in hydroxylation level seen in shaker ask cultures and allow
high-level production, and whether the heterotrimeric recombinant type I collagen molecules would
form native-type brils in vitro.
Copyright # 2001 John Wiley & Sons, Ltd.

Materials and methods

Expression vector constructs and generation
of recombinant P. pastoris strains
The vectors pBLARGIX, pBLARGSX and
pBLADEIX, and the P. pastoris strain yJC300
(his4, arg4, ade1) were gifts from Dr James Cregg.
All the recombinant strains were generated by
electroporation, as described in the Manual of
Methods for Expression of Recombinant Proteins in
Pichia pastoris (Invitrogen). The recombinant
strains had the methanol utilization plus phenotype.

Proa1(I) and PCa1(I) strains

The strain a/PDI-aMF expressing 4-PH has been
described previously (Vuorela et al., 1997). To
generate pPICZBproa1(I) (Vuorela et al., 1999),
an EcoRIScaI fragment was digested from
pVL1392proa1(I) (Myllyharju et al., 1997), a
second fragment extending from the internal ScaI
site to the translation stop codon followed by a
MfeI site was synthesized by PCR, and these
fragments were co-ligated into the EcoRI site of
pPICZB (Invitrogen). To delete the sequence
encoding the N propeptide, two fragments were
generated by PCR with pVL1392proa1(I) as a
template, the rst extending from the pVL1392
PstI site to the codon for the last amino acid of the
signal peptide, and the second from the codon for
the rst amino acid of the N telopeptide to an
internal DraIII site. These fragments were ligated
into PstIDraIII-digested pVL1392proa1(I), and an
EcoRI fragment was cut from this plasmid and
ligated into pPICZBproa1(I) digested with EcoRI
to generate pPICZBpCa1(I). The pPICZBproa1(I)
and pPICZBpCa1(I) constructs were linearized with
PmeI and transformed separately into the a/PDIaMF strain, thus generating the strains Proa1(I)
and PCa1(I), respectively (Table 1).

Proa2(I) and PCa2(I) strains

A new strain expressing 4-PH was made by ligating
the coding region of the a subunit cDNA clone
PA59 as a SmaI fragment (Vuorela et al., 1997) into
the EcoRI site of pBLARGIX, and co-transforming
pBLARGIXa and pPIC9PDI (Vuorela et al., 1997),
linearized with HincII and StuI, respectively, into
the yJC300 (his4, arg4, ade1) strain. To generate
pBLADEIXproa2(I), the BglIIBamHI proa2(I)
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High-level production of human type I collagen in P. pastoris

799

Table 1. Recombinant Pichia strains

Strain

Expression vectors

Selection

Polypeptides expresseda

Proa1(I)
Proa2(I)
Proa1(I)+Proa2(I)

pPICZBproa1(I), pARG815a, pPIC9PDI

pBLADEIXproa2(I), pBLARGIXa, pPIC9PDI
pPICZBproa1(I), pBLADEIXproa2(I),
pBLARGIXa, pPIC9PDI
PAO815proa2(I)/proa1(I),
pBLADEIXa, pBLARGSXPDI
pPICZBpCa1(I), pARG815a, pPIC9PDI
pBLADEIXpCa2(I), pBLARGIXa, pPIC9PDI
pPICZBpCa1(I), pBLADEIXpCa2(I),
pBLARGIXa, PPIC9PDI

Zeo+b, Arg+, His+

Ade+, Arg+, His+
Zeo+, Ade+, Arg+, His+

proa1(I)chain, 4-PH a subunit, PDI

proa2(I)chain, 4-PH a subunit, PDI
proa1(I) and proa2(I) chains,
4-PH a subunit, PDI
proa1(I) and proa2(I) chains,
4-PH a subunit, PDI
pCa1(I)chain, 4-PH a subunit, PDI
pCa2(I)chain, 4-PH a subunit, PDI
pCa1(I) and pCa2(I) chains,
4-PH a subunit, PDI

Proa2(I)/Proa1(I)
PCa1(I)
PCa2(I)
PCa1(I)+PCa2(I)

His+, Ade+, Arg+

Zeo+, Arg+, His+
Ade+, Arg+, His+
Zeo+, Ade+,
Arg+, His+

The PDI polypeptide that acts as the b subunit of 4-PH has the Saccharomyces cerevisiae a mating factor (aMF) prepropeptide in all strains,
whereas all other polypeptides have their own signal sequences.
b
Zeocin resistance.
a

cDNA obtained from pSP72proa2(I) (Myllyharju

et al., 1997) was cloned into the BamHI site of
pMALc2 (New England BioLabs), an EcoRISacI
fragment was digested from this construct, a second
fragment extending from the internal SacI site to
the translation stop codon followed by a MfeI site
was synthesized by PCR, and these fragments were
co-ligated into the EcoRI site of pBLADEIX. To
delete the sequence encoding the N propeptide,
two fragments were generated by PCR with
pSP72proa2(I) as a template, the rst extending
from the BglII site to the codon for the last amino
acid of the signal peptide, and the second from the
codon for the rst amino acid of the N telopeptide
to an internal SacII site. These fragments were
ligated into BglIISacII digested pSP72proa2(I), the
BglIIBamHI pCa2(I) cDNA obtained from this
construct was cloned into the BamHI site of
pMALc2, and an EcoRI fragment was cut from
this and ligated into pBLADEIXproa2(I) digested
with EcoRI to generate pBLADEIXpCa2(I). The
pBLADEIXproa2(I) and pBLADEIXpCa2(I) were
linearized with SpeI, transformed separately into
the strain expressing 4-PH, and the strains were
named Proa2(I) and PCa2(I), respectively (Table 1).

Proa1(I)+Proa2(I) and PCa1(I)+PCa2(I)

strains
The pPICZBproa1(I) and pPICZBpCa1(I) constructs linearized with PmeI were transformed into
the Proa2(I) and PCa2(I) strains, respectively, to
generate the strains Proa1(I)+Proa2(I) and
PCa1(I)+PCa2(I) (Table 1).
Copyright # 2001 John Wiley & Sons, Ltd.

Proa2(I)/Proa1(I) strain
A third strain expressing 4-PH was rst generated.
The coding region of the a subunit cDNA PA59
was ligated as a SmaI fragment (Vuorela et al.,
1997) into the EcoRI site of pBLADEIX and an
internal XbaI site in the cDNA was removed by
site-directed mutagenesis (Stratagene). A PDI
cDNA extending from the codon for the rst
amino acid after the signal peptide (Vuorela et al.,
1997) was ligated into the EcoRI site following the
S. cerevisiae a mating factor prepro sequence
in
pBLARGSX.
The
pBLADEIXa
and
pBLARGSXPDI, linearized with XbaI and BglII,
respectively, were co-transformed into the yJC300
(his4, arg4, ade1) strain. To generate a proa2(I)/
proa1(I) double expression vector, the cDNAs were
rst cloned separately into the EcoRI site of pAO815
(Invitrogen), as described for pPICZBproa1(I) and
pBLADEIXproa2(I). The proa1(I) expression cassette was cut from pAO815proa1(I) in two fragments, BglIIBamHI and BamHIBamHI, and
cloned downstream of the proa2(I) cassette into
pAO815proa2(I) digested with BamHI to generate
pAO815proa2(I)/proa1(I). This construct was linearized with SalI and transformed into the strain
expressing 4-PH described above. The strain was
named Proa2(I)/Proa1(I) (Table 1).

Growth and induction of P. pastoris in shaker

asks
Cells were grown in 25 ml shaker ask cultures in a
buffered glycerol complex medium (BMGY,
pH 6.0) with 1 g/l yeast extract and 2 g/l peptone.
Expression was induced in a buffered methanol
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800

complex medium (BMMY, pH 6.0), and methanol

was added every 12 h to a nal concentration of
0.5%. Amino acids were added up to 100 mg/l as
required.

Analysis of the recombinant collagen

Cells were harvested after a 60 h methanol induction at 30uC, washed once and suspended in a cold
(4uC) 5% glycerol, 1 mM Pefabloc SC and 50 mM
sodium phosphate buffer, pH 7.4. The cells were
broken by vortexing with glass beads, and the lysate
was centrifuged at 10 000rg for 30 min. Protein
concentrations were determined using the Bio-Rad
Protein Assay (Bio-Rad). Aliquots of the soluble
fractions of the cell lysates were analysed by
SDSPAGE under reducing conditions, followed
by Western blotting with polyclonal antibodies 1675
and 1669 (FibroGen), recognizing the N propeptide
of human proa1(I) and the C propeptide of human
proa2(I) chains, respectively, or a monoclonal antibody 95D1A (Snellman et al., 2000) recognizing the
collagenous regions of various collagen chains.
Further aliquots of the soluble fraction were
analysed by radioimmunoassays for the trimeric N
propeptide (PINPRIA) or C propeptide (PICP
RIA) of human type I procollagen (Farmos
Diagnostica), and others were digested with pepsin
for 2 h at 22uC and analysed by SDSPAGE
followed by silver staining. The amounts of the
collagen chains were estimated by densitometry of
the silver-stained bands using a GS-710 Calibrated
Imaging Densitometer (BioRad). 4-PH activity was
assayed by a method based on the hydroxylationcoupled decarboxylation of 2-oxo[1-14C]glutarate
(Kivirikko and Myllyla, 1982).

Fermentation of the recombinant P. pastoris

strains
Fermentation was performed according to Invitrogen guidelines in a Biostat C bench-top fermenter
(B. Braun) equipped with a 2 l water-jacketed
stainless steel vessel, an O2 supply unit, and
microprocessor control of pH, dissolved oxygen,
agitation, temperature and nutrient feed, and
electronic foam control. The vessel containing 1 l
medium composed of Fermentation Basal Salts and
4% glycerol was sterilized for 30 min at 122uC, after
which 2.4 ml/l trace salts (Invitrogen, PTM1) and
cells obtained from a 100 ml start-up culture in
BMGY were added aseptically. Agitation was
Copyright # 2001 John Wiley & Sons, Ltd.

M. Nokelainen et al.

maintained at 800 rpm, temperature at 30uC and

O2 level at 30%, and pH 5.0 was controlled with
25% ammonium hydroxide. The glycerol batch
phase was continued until the glycerol was completely consumed (24 h), and the glycerol-feed phase
was initiated by continuously feeding a medium
containing 50% (w/v) glycerol and 12 ml/l PTM1
trace salts at a rate of 18 ml/h/l until the amount of
glycerol fed in was 100200 g/l. The methanol-feed
phase was started by feeding methanol containing
12 ml/l PTM1 trace salts at a rate of 2 g/h/l for 18 h,
the feed was increased to 4 g/h/l for 3 h, 6 g/h/l for
3 h, and nally 67 g/h/l for the rest of the phase,
which lasted 56 days. The cells were harvested and
centrifuged at 2 500rg for 10 min at 4uC and
frozen at x20uC.

Purication of the recombinant collagen

The cells were suspended in 0.1 M HCl, broken by
agitation with glass beads using a Mini-Beater
(Biospec Products), and the lysate was ltered
through a Buchner Funnel without lter paper
(Polypropylene, Nalgene). The ltrate was digested
with a nal concentration of 0.2 mg/ml of pepsin
for 18 h at 4uC, centrifuged at 10 000rg for 30 min,
and the collagen was precipitated by adding solid
NaCl to a nal concentration of 3 M and acetic acid
to 0.5 M. The pellet obtained by centrifugation was
dissolved in 0.1 M HCl, and the collagen was
reprecipitated at 4uC for 1 h by adjusting the pH
to 7.4 and adding NaCl to a nal concentration of
3 M. The pellet obtained was redissolved in 0.1 M
HCl, dialysed against 1 M acetic acid, and chromatographed on a Sephacryl S-500HR column in the
KTA explorer system (Amersham Pharmacia
A
Biotech). Amino acid analyses and N-terminal
sequencing were performed in an Applied Biosystems 421A amino acid analyser and in an Applied
Biosystems 477A pulse-liquid protein sequencer,
respectively.

In vitro bril formation and electron

microscopy
The collagens were adjusted to various concentrations, and the self-assembly followed a modication
of a described method (Williams et al., 1978)
employing 30 mM K2HPO4 and 135 mM NaCl,
pH 7.4. Fibril formation was triggered by increasing
the temperature to 34uC, optical density was
monitored at 313 nm, the samples were transferred
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High-level production of human type I collagen in P. pastoris

after 1200 min to Formvar-coated copper grids

using a micropipette, and the brils were allowed
to settle for 30 min. The buffer was then drained
cautiously, and three washing steps were performed.
The brils were stained for 2 min with freshly
prepared 1% phosphotungstate, pH 7.4, washed
three times and dried. The grids were examined
using a Zeiss EM 109 electron microscope, and the
bril widths were measured with Scion Image
program (Scion Corporation, USA).

Results
Generation of recombinant P. pastoris strains
co-expressing human type I procollagen and
4-PH, and analysis of the recombinant proteins
The strains Proa1(I), Proa2(I), Proa1(I)+Proa2(I)
and Proa2(I)/Proa1(I) that expressed the corresponding polypeptide chains of human type I
procollagen and human 4-PH (Table 1) were generated as described in Materials and methods. The
recombinant strains were cultured in BMGY
medium, and expression was induced in BMMY
medium, methanol being added every 12 h. The
cells were harvested and broken 60 h after induction, and aliquots of the soluble fraction of the cell
extracts were analysed by SDSPAGE followed by
Western blotting. Full-length proa1(I) chains were
seen in immunoblots of the strains Proa1(I),
Proa1(I)+Proa2(I) and Proa2(I)/Proa1(I) (Figure 1A,
lanes 1, 3 and 4), and full-length proa2(I) chains
were seen in those of the Proa2(I), Proa1(I)+

801

Proa2(I) and Proa2(I)/Proa1(I) strains (Figure 1A,

lanes 68).
The levels of expression in at least 10 transformants of each strain were measured with a radioimmunoassay for the trimeric N propeptide. The
mean expression level of the type I procollagen
homotrimer was 110 ng/100 mg total protein (range
45185 ng/100 mg) and that of the heterotrimer
60 ng/100 mg (range 25135 ng/100 mg). No differences in the levels of expression were seen between
the Proa1(I)+Proa2(I) and Proa2(I)/Proa1(I)
strains.
The presence of triple-helical procollagen molecules was assayed by digesting aliquots of the cell
extracts with pepsin. The triple helix of collagens is
resistant to pepsin, while non-triple-helical proa
chains and the propeptides of procollagen molecules are digested (Bruckner and Prockop, 1981). A
single pepsin-resistant polypeptide corresponding to
the a1(I) chains was seen in samples from
the Proa1(I) strain in silver-stained SDSPAGE
(Figure 1B, lane 1), whereas no pepsin-resistant
polypeptide was seen in samples from the Proa2(I)
strain (Figure 1B, lane 2). Samples from the
Proa1(I)+Proa2(I) and Proa2(I)/Proa1(I) strains
had two pepsin-resistant polypeptides corresponding to the a1(I) and a2(I) chains. Densitometry of
the bands in six individual samples indicated that
the a1(I) to a2(I) chain ratio was 1.92t0.13 (SD) to
1 (Figure 1B, lanes 3 and 4). Thus, all the pepsinresistant a2(I) chains must have been present in the
heterotrimeric molecules and, as the ratio of the
a1(I) and a2(I) chains did not exceed 2 : 1, essentially

Figure 1. Expression of the recombinant human type I procollagen homotrimers and heterotrimers in P. pastoris. (A) 50 mg
samples of the soluble fractions of the cell lysates from the strains Proa1(I) (lanes 1 and 5), Proa2(I) (lanes 2 and 6),
Proa1(I)+Proa2(I) (lanes 3 and 7) and Proa2(I)/Proa1(I) (lanes 4 and 8) were analysed by SDSPAGE under reducing
conditions followed by Western blotting, using antibodies against the N propeptide of the human proa1(I) chain (lanes 14)
and C propeptide of the human proa2(I) chain (lanes 58). The arrows indicate the proa1(I) and proa2(I) chains. The lines on
the left indicate the positions of the molecular weight markers (kDa). (B) 20 ml aliquots of pepsin-treated samples from the
strains Proa1(I) (lane 1), Proa2(I) (lane 2), Proa1(I)+Proa2(I) (lane 3) and Proa2(I)/Proa1(I) (lane 4) were analysed by
SDSPAGE under reducing conditions followed by silver staining. The arrows indicate the a1(I) and a2(I) chains
Copyright # 2001 John Wiley & Sons, Ltd.

Yeast 2001; 18: 797806.

802

all the a1(I) chains must likewise have been present

in heterotrimers.

Analysis of the expression of recombinant

human type I pCcollagen in Pichia
In order to study whether type I pCa chains (chains
lacking their N propeptide) form triple-helical
pCcollagen molecules, the strains PCa1(I) and
PCa1(I)+PCa2(I) (Table 1) were generated. These
strains were cultured, induced and broken as above,
and the soluble fractions of the cell extracts were
analysed by SDSPAGE, followed by Western
blotting using an antibody against the collagenous
regions of various collagens. PCa1(I) chains were
seen in the immunoblots of the strains PCa1(I) and
PCa1(I)+PCa2(I) (Figure 2A, lanes 3 and 4), and
pCa2(I) chains in those of the PCa1(I)+PCa2(I)
strain (Figure 2A, lane 4). In the case of the pepsindigested samples, a polypeptide corresponding to
the a1(I) chains was seen in the PCa1(I) strain
(Figure 2B, lane 1), and two polypeptides with a
2 : 1 ratio corresponding to the a1(I) and a2(I)
chains were seen in the PCa1(I)+PCa2(I) strain
(Figure 2B, lane 2). The type I pCa chains therefore
became efciently assembled into triple-helical
molecules. The levels of expression of type I
pCcollagen, when measured with a radioimmunoassay for the trimeric C propeptide, were found to
be 1.53-fold relative to those of the corresponding
type I procollagen molecules. The mean level of
expression of the pCcollagen homotrimer was
160 ng/100 mg total protein (range 85265 ng/
100 mg) and that of the pCcollagen heterotrimer
150 ng/100 mg (range 85275 ng/100 mg).

M. Nokelainen et al.

Production of type I collagen using a

fermenter and characterization of the
recombinant protein
The Proa1(I), Proa1(I)+Proa2(I), PCa1(I) and
PCa1(I)+PCa2(I) strains were cultured and
induced in a 2 l fermenter equipped with an O2
supply system. The cells were harvested 6 days after
induction, and the levels of expression of the type I
procollagen and pCcollagen homotrimers and
heterotrimers were measured as above. These
levels were in the range 200500 mg/l, the highest
levels being seen with the pCcollagen trimers.
The recombinant collagens were puried by
digesting the cell lysates with pepsin, which converts
the procollagen or pCcollagen to collagen and
digests most of the non-collagenous proteins, this
being followed by selective salt precipitation and gel
ltration. The recombinant collagen trimers were
essentially pure when analysed by SDSPAGE
followed by Coomassie blue staining (Figure 3).
Amino acid analyses showed that the 4hydroxyproline contents of the puried homotrimers and heterotrimers were identical to those of
the corresponding non-recombinant proteins, the
only difference between the recombinant and nonrecombinant collagens being the lack of hydroxylysine and the corresponding increase in the lysine
content in the recombinant molecules (Table 2). Nterminal sequencing of the a chains derived from
the pCcollagen homotrimers and heterotrimers
indicated that in most cases the pepsin digestion
had removed no amino acid from the N terminus of
the a1(I) and a2(I) chains, and only minor populations of the chains had lost ve residues or one
residue, respectively (details not shown).

Figure 2. Expression of the recombinant human type I pCcollagen homotrimers and heterotrimers in P. pastoris. (A) 50 mg
samples of the soluble fractions of the cell lysates from the strain expressing only 4-PH (lane 1) and the strains Proa1(I) (lane
2), PCa1(I) (lane 3) and PCa1(I)+PCa2(I) (lane 4) were analysed by SDSPAGE under reducing conditions followed by
Western blotting, using an antibody against the collagenous region of various collagens. The arrows indicate the proa1(I),
pCa1(I) and pCa2(I) chains. (B) 20 ml aliquots of pepsin-treated samples from the strains PCa1(I) (lane 1) and
PCa1(I)+PCa2(I) (lane 2) were analysed by SDSPAGE under reducing conditions followed by silver staining. The arrows
indicate the a1(I) and a2(I) chains
Copyright # 2001 John Wiley & Sons, Ltd.

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High-level production of human type I collagen in P. pastoris

Figure 3. Analysis of puried recombinant human type I

collagens expressed in P. pastoris. Recombinant human type I
collagen homotrimers and heterotrimers were puried from
the strains Proa1(I) (lane 1), Proa1(I)+Proa2(I) (lane 2),
PCa1(I) (lane 3) and PCa1(I)+PCa2(I) (lane 4) and analysed
by SDSPAGE followed by Coomassie blue staining. The
arrows indicate the a1(I) and a2(I) chains

Formation of recombinant type I collagen brils

was studied by incubating the collagen solution at
34uC in vitro at pH 7.4. The resulting brils formed
showed the banding pattern characteristic of collagen brils in electron microscopy and were
indistinguishable from those formed by nonrecombinant type I collagen (Figure 4).

803

Figure 4. Electron micrographs of brils formed in vitro by

the recombinant type I collagen

Discussion
The data presented here demonstrate that it is
possible to integrate four genes into the genome of
P. pastoris and successfully co-express all four
polypeptides. The two types of 4-PH subunit

Table 2. Amino acid analysis of the puried recombinant human type I collagen homotrimer and heterotrimer

Amino acid

Recombinant
human type I
collagen homotrimera
(residues/1000)

Non-recombinant
human type I
collagen homotrimerb
(residues/1000)

Recombinant human
type I collagen heterotrimera
(residues/1000)

Non-recombinant
human type I
collagen heterotrimerb
(residues/1000)

Aspartic acid
Glutamic acid
4-Hydroxyproline
Serine
Glycine
Histidine
Arginine
Threonine
Alanine
Proline
Valine
Methionine
Tyrosine
Isoleusine
Leucine
Hydroxylysine
Phenylalanine
Lysine

46t4.3
77t4.4
110t2.2
35t1.6
314t5.4
4.8t0.5
50t1.3
19t0.8
120t13.3
122t2.9
21t2.2
NDc
ND
9t0.1
20t2.8
0
11t1.0
33t1.6

42
73
108
34
333
3
50
16
115
124
21
7
1
6
19
9
12
26

40t5.9
71t0.3
99t5.7
31t3.6
315t19
6.9t1.5
53t1.7
19t3.2
123t1.5
125t14
25t0.4
ND
ND
9t1.1
23t1.7
0
10t1.2
34t1.9

43
71
103
33
335
6
50
17
111
120
26
6
2
9
23
10
12
23

The values are given as meanstSD. n=35.

Miller and Gay (1982).
c
ND, not determined.
b

Copyright # 2001 John Wiley & Sons, Ltd.

Yeast 2001; 18: 797806.

804

became assembled into an active a2b2 tetramer that

effectively hydroxylated the two types of proa
chain, and the proa1(I) and proa2(I) chains
formed heterotrimeric molecules with the correct
2 : 1 chain ratio. In addition, the recombinant type I
collagen molecules produced from the procollagen
molecules by pepsin digestion formed native-type
brils in vitro. The procedure reported here may
represent the most complex system described so far
for the production and processing of any protein in
Pichia. After the completion of the experiments
reported here, Toman et al. (2000) reported the
production of human type I collagen in Saccharomyces cerevisiae. 4-PH was found to hydroxylate
the two types of proa chain also in the Saccharomyces system, but the level of hydroxylation
obtained was only about 80% of that found in vivo.
Pepsin digestion is known to remove several
residues from the short non-triple-helical regions
present in the N and C termini of the a chains
(Kielty et al., 1993; Kuznetsova and Leikin, 1999).
These regions are called telopeptides and are
believed to play a role in bril formation (Kielty
et al., 1993; Prockop and Kivirikko, 1995; Kadler
et al., 1996). However, a recent study (Kuznetsova
and Leikin, 1999) indicates that this role is only
seen in the kinetics of the process, whereas even
molecules essentially lacking their entire telopeptides form brils that are identical to those formed
by the full-length molecules. The present data
indicating that our recombinant type I collagen
molecules produced from procollagen by pepsin
treatment formed native-type brils are in agreement with this conclusion.
The N propeptides are believed to play no role in
the assembly of the procollagen molecule within the
endoplasmic reticulum (Prockop and Kivirikko,
1995). As pepsin is an endopeptidase, we considered
it possible that this enzyme might digest the N
termini of the a chains less effectively in molecules
lacking the N propeptides. We therefore generated
expression constructs in which sequences encoding
the signal peptide were directly followed by those
encoding the N telopeptide. The pCa1(I) and
pCa2(I) chains produced were found to form
heterotrimeric pCcollagen molecules with the correct 2 : 1 chain ratio and, interestingly, the levels of
expression of the pCcollagen homotrimers and
heterotrimers were found to be 1.53-fold relative
to those of the corresponding procollagen trimers.
N-terminal sequencing of the a chains in molecules
Copyright # 2001 John Wiley & Sons, Ltd.

M. Nokelainen et al.

derived from pCcollagen indicated that most of the

a1(I) and a2(I) chains indeed had N termini that
were identical to those of the corresponding nonrecombinant chains. The use of the expression
constructs for the pCa chains also eliminates the
possibility that in the case of incomplete pepsin
digestion the a chains may contain some amino acid
residues that belong to the C-terminal end of the N
propeptide. We did not study whether it was also
possible to omit the sequences encoding the C
propeptides from the expression constructs, as these
propeptides are known to be essential for correct
chain recognition and chain alignment and to play a
crucial role in chain assembly (Kielty et al., 1993;
Prockop and Kivirikko, 1995; Kadler et al., 1996;
Lees et al., 1997).
As the Km of O2 in the 4-PH reaction is as high as
40 mM (Kivirikko and Pihlajaniemi, 1998), we have
thought it likely that the O2 concentration within
the lumen of the endoplasmic reticulum of the yeast
cells is rate-limiting for hydroxylation when a
collagen is produced in a P. pastoris strain expressing 4-PH in shaker ask cultures (Vuorela et al.,
1997). We have therefore suggested that the
deciency in the hydroxylation of the recombinant
type III collagen seen under such conditions might
disappear when the protein was produced in a
fermenter equipped with an O2 supply system
(Vuorela et al., 1997). The present data indicate
that the 4-hydroxyproline contents of the recombinant type I collagen molecules produced in the 2 l
fermenter were identical to those of the corresponding non-recombinant proteins. In addition, we have
recently found that the 4-hydroxyproline contents
of recombinant type III collagen molecules produced in the fermenter are likewise identical to
those of the corresponding non-recombinant protein (M. Nokelainen, A. Vuorela, K.I. Kivirikko,
J. Myllyharju, unpublished observation). Use of
the fermenter also made it possible to grow the
P. pastoris strains to high cell densities and this
markedly increased the expression levels per culture
volume, as has been shown previously for many
other proteins in Pichia (Cregg et al., 1993;
Cereghino and Cregg, 2000). It should be noted
that all the experiments reported here were performed with single-copy integrants and using
human cDNAs with their authentic sequences.
Many previous studies have demonstrated that the
expression levels of various proteins in P. pastoris
increase markedly with the numbers of DNA
Yeast 2001; 18: 797806.

High-level production of human type I collagen in P. pastoris

copies, at least up to 3050 copies (Cregg et al.,

1993; Scorer et al., 1994; Cereghino and Cregg,
2000). Furthermore, it has been pointed out that the
sequences encoding the human type I procollagen
chains differ very markedly in their codon usages
from those encoding various yeast proteins (Werten
et al., 1999). It would thus seem possible to
optimize the current expression level, which is up
to 500 mg/l, for the production of very large
amounts of recombinant human type I collagen
for various medical applications.

Acknowledgements
We thank Dr James Cregg, Keck Graduate Institute of
Applied Life Sciences, for the gift of the pBLARGIX,
pBLARGSX and pBLADEIX vectors and the P. pastoris
strain yJC300, and Raija Juntunen, Anne Kokko, Eeva
Lehtimaki, Minna Siurua and Tanja Vaisanen for their
expert technical assistance. This work was supported by
grants from the Health Science Council of the Academy of
Finland, the Finnish Centre of Excellence Programme
20002005 (44843), from the European Commission (BIO4CT96-0537), from the National Institutes of Health (R01
AR45879), from FibroGen Inc. (South San Francisco, CA),
from the Research and Science Foundation of Farmos, and
from the Jenny and Antti Wihuri Foundation.

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