THE JOURNAL OF BIOLOGICAL CHEMISTRY

2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 30, Issue of July 28, pp. 2330323309, 2000
Printed in U.S.A.

Production of Recombinant Human Type I Procollagen Trimers

Using a Four-gene Expression System in the Yeast
Saccharomyces cerevisiae*
Received for publication, March 17, 2000, and in revised form, May 5, 2000
Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M002284200

P. David Toman, George Chisholm, Hugh McMullin, Lynne M. Giere, David R. Olsen,
Robert J. Kovach, Scott D. Leigh, Bryant E. Fong, Robert Chang, Gregory A. Daniels,
Richard A. Berg, and Ronald A. Hitzeman
From Cohesion Technologies, Palo Alto, California 94303 and Genotypes Incorporated,
South San Francisco, California 94080

The expression of stable recombinant human collagen

requires an expression system capable of post-translational modifications and assembly of the procollagen
polypeptides. Two genes were expressed in the yeast
Saccharomyces cerevisiae to produce both propeptide
chains that constitute human type I procollagen. Two
additional genes were expressed coding for the subunits
of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the
collagen molecule. Type I procollagen was produced as a
stable heterotrimeric helix similar to type I procollagen
produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain
high expression levels of type I procollagen as heatstable heterotrimers in Saccharomyces. Expression of
these four genes was sufficient for correct assembly and
processing of type I procollagen in a eucaryotic system
that does not produce collagen.

Collagen is the single most abundant protein found in animals. It is found in all animals, including sponges. It is not
expressed in yeast. In mammals, it is expressed in most tissues
and plays both a structural as well as a signaling role in the
development, maintenance, and repair of tissues and organs.
More than 30 gene products compose the collagen family of
molecules (1). Procollagens have several features and require
numerous steps for production of functional molecules, including post-translational modifications (2). Key features in the
collagen family are the formation of a triple helix composed of
three polypeptide chains and the post-translational modification of proline residues to hydroxyproline, which provides stability of the triple helix against thermal denaturation and
unfolding (Tm)1 at the animals body temperature (3). The
content of proline and hydroxyproline is correlated with the
temperature of an animals environment (4). The triple helical
domain of procollagen consists of -(GXY)n- repeats, where X
and/or Y is frequently proline or hydroxyproline in the mature
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
To whom correspondence should be addressed: Cohesion Technologies, 2500 Faber Place, Palo Alto, CA 94303. Tel.: 650-320-5595; Fax:
650-320-5511; E-mail: dtoman@cson.com.
1
The abbreviations used are: Tm, melting temperature; PH, prolyl
hydroxylase -subunit; PDI, protein-disulfide isomerase; PCR, polymerase chain reaction; bp, base pair(s); HSA, human serum albumin;
PIPES, 1,4-piperazinediethanesulfonic acid.
This paper is available on line at http://www.jbc.org

molecule. Prolyl 4-hydroxylase, an 22 tetrameric enzyme

composed of the prolyl hydroxylase -subunit (PH) and the
protein-disulfide isomerase (PDI) subunit in higher eucaryotes,
is the enzyme that modifies proline residues to hydroxyproline.
Additional steps for procollagen production include carbohydrate attachment, folding into a triple helix, secretion into the
extracellular matrix, and cleavage by specific proteases to remove the propeptide domains to form mature collagen helices.
A C-terminal non-helical propeptide facilitates the assembly of
trimeric collagen molecules, leading to helix formation (5); the
N-terminal propeptide may limit fiber diameter (6). The association and folding steps of three polypeptide chains that compose the triple helix potentially require chaperone functions in
the endoplasmic reticulum, with PDI (7) and Hsp47 (8) as two
proteins that have been implicated in the assembly of a procollagen trimer.
A fundamental question regarding collagen biosynthesis is
which genes are essential for the expression of collagen in cells
and which are nonessential. Expression of recombinant collagen has been performed using mammalian, baculoviral, and
transgenic systems. Single procollagen genes were expressed in
mammalian cells to produce homotrimeric type I procollagen
(9), type II procollagen (10), and homotrimeric type V collagen
(11). In baculovirus, prolyl hydroxylase was transfected and
shown to be a functional enzyme (12). Subsequently, type I and
III procollagens were transfected and expressed and were
shown to be capable of modification by prolyl hydroxylase (13
15). Recently, homotrimeric type I procollagen and an engineered form of 2(I) procollagen have been expressed in the
milk of transgenic mice (16, 17). In contrast, no report of
procollagen expression and assembly has been published using
a bacterial expression system.
The yeast Pichia pastoris was first engineered to express
prolyl hydroxylase and subsequently shown to produce functional type III procollagen if the gene for type III procollagen
was introduced (18, 19). Like Saccharomyces, Pichia contains
endogenous PDI, but not PH, and it does not synthesize procollagen. It was therefore a useful system to test the requirements for genes to produce type III procollagen. In this system,
the type III procollagen gene and the two genes for prolyl
hydroxylase were sufficient to produce stable type III procollagen molecules. However, Saccharomyces is evolutionarily diverse from Pichia. Furthermore, type I procollagen is composed
of polypeptides generated from two distinct genes to form an
(1)22 structure, whereas type II and III procollagens require
only one gene product to form an (1)3 structure.
To our knowledge, this is the first report to describe a multigene system in Saccharomyces that results in both the assem-

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23304

Type I Procollagen Expression in Yeast

bly and non-native post-translational modification of a multimeric protein to produce a functional heterologous molecule. A
total of four gene products were required in Saccharomyces to
generate a thermally stable triple helical type I procollagen:
two genes that code for the polypeptide chains of type I procollagen and two additional genes that code for the subunits of
prolyl hydroxylase. No other added genes were required to
produce a functional procollagen. We further optimized our
expression system at the molecular level, but also optimized
the addition of medium components to significantly increase
the level of expression of type I procollagen in Saccharomyces.
EXPERIMENTAL PROCEDURES

Plasmid ConstructionsThe precursor plasmid pGET100 and plasmid pGET150, which contains the GAL1/GAL10 dual promoter and is
the base plasmid for other constructions, were made as follows. YEp9T,
containing yeast (2 origin, FLP1 gene terminator in 2 DNA, and the
yeast TRP1 gene) and bacterial (pBR322 functions) sequences (20), was
modified between an NdeI site in the 2 DNA and a second NdeI site
near the origin of pBR322 with the polylinker sequence (NdeI)-PvuIIApaI-BglII-ClaI-NheI-XhoI-EcoRI-BamHI-AflII-NotI-(NdeI) to create
pGET100 (PvuII site closest to 2 DNA). Genomic DNA from yeast
strain S1799D (MAT trp5 his4 ade6 gal2) was used as the template for
PCR with primers (based on sequence information (21)) containing
BamHI placed at 6 of the GAL1 promoter side and EcoRI placed at 1
of the GAL10 promoter side. The 687-bp EcoRI/BamHI GAL1/GAL10
product was subcloned into pUC. This fragment was then placed into
the EcoRI/BamHI sites of pGET100 to make pGET150. The specific
structure of circular plasmid pGET150 is as follows: ApR (ampicillin
resistance gene)-yeast TRP1-yeast 2 origin-FLP1 terminator-PvuIIpolylinker-EcoRI-GAL10 promoter/GAL1 promoter-BamHI-AflII-NotI(NdeI)-SapI-Escherichia coli origin of replication-ApR. Both sides of this
dual promoter are inducible with galactose and repressible by glucose.
Plasmid pGET333, expressing human 1(I) preprocollagen, was constructed by cloning an SspI/XbaI fragment containing the human 1(I)
preprocollagen cDNA (22) coding region between the PvuII and NheI
sites in the polylinker of pGET150. To express human 1(I) procollagen
using other secretion signals known to work well in yeast, a SalI site
was introduced at the pre/pro junction (just upstream of amino acid 23
in preprocollagen), removing the 22-amino acid presequence using PCR.
This site was used to fuse two secretion signals to the 1(I) procollagen
gene using the artificial SalI site and the EcoRI site adjacent to the
GAL promoter in pGET333. Plasmids pGET323 and pGET335 contain
the prepro-human serum albumin (HSA) (23) secretion signal and the
yeast prepro--factor (24) secretion signal, respectively. The prepro-factor signal was isolated using PCR, whereas the prepro-HSA signal
was constructed from synthetic oligonucleotides. Both sequences were
isolated as EcoRI/SalI fragments with the SalI site containing the
Arg-Arg KEX2 protease cleavage site (23) at the end of these prosequences to give authentic procollagen protein.
The general structure of all other plasmids is as follows: ApR-yeast
TRP1-2 origin-FLP1 terminator-PvuII-SspI-1(I) preprocollagenXbaI-NheI-XhoI-EcoRI-GAL10 promoter/GAL1 promoter-BamHI-AflII2(I) preprocollagen-3-phosphoglycerate kinase gene terminatorNotI PMA1 promoter-yeast invertase secretion signal-PDI geneADH1 terminator-NotI-(NdeI)-SapI 3-phosphoglycerate kinase gene
promoter-PH-GAL10 terminator-SapI-E. coli origin of replicationApR. PMA1 and ADH1 refer to the plasmid membrane ATPase 1 gene
and the alcohol dehydrogenase 1 gene, respectively, isolated from yeast.
The full-length cDNA for human 2(I) preprocollagen has been described (25). The PDI gene used was from either chicken (26) or human
(27) utilizing the yeast invertase secretion signal (23), replacing the
first 22 amino acids of the chicken PDI gene. The PH gene cDNA was
from chicken (28) or human (29). The 3-phosphoglycerate kinase gene
promoter (828 bp from the natural ClaI site to the introduced EcoRI site
upstream of ATG), PMA1 promoter (the 939-bp fragment from the
natural HindIII site to the introduced EcoRI site upstream of ATG),
3-phosphoglycerate kinase gene terminator (the 301-bp BamHI/SmaI
fragment to the HindIII/NotI fragment using PCR to add NotI to HindIII and to make sites devoid of the 3-phosphoglycerate kinase structural gene), ADH1 terminator (the natural 330-bp HindIII fragment),
and GAL10 terminator (the 360-bp BamHI/SphI fragment) elements
were originally isolated by PCR based on sequences and references in
the Saccharomyces Genome Data Base (Department of Genetics, School
of Medicine, Stanford University). Some ends and junctions were created using synthetic oligonucleotides.

Plasmid pGET737 contains only human 1(I) and 2(I) preprocollagen genes as described above. The human or chicken PDI and PH
expression units were added to plasmid pGET737 as NotI or SapI
fragments, respectively, to create plasmids pGET837 (chicken PDI and
human PH), pGET901 (chicken PDI and PH), and pGET903 (human
PDI and PH). Strain GY5382 contains integrated chicken PH and
PDI cDNA expression units in the yeast TRP1 locus, resulting in a
trp1 strain that expresses both of these genes under the control of the
GAL10 promoter/GAL1 promoter elements. The EcoRI/PstI fragment of
the TRP1 gene (30) was cloned into the pBluescript II SK vector. An
EcoRI/BamHI fragment was then placed into pBR322 with subsequent
deletion of the MfeI/BstXI fragment within the TRP1 structural gene
and replacement with the polylinker (MfeI)-NotI-BglII-XhoI-(BstXI).
The HindIII fragment containing the URA3 yeast gene (31) was converted to a SalI fragment and placed into the above XhoI site. A NotI
fragment containing the ADH1 terminator-chicken PDI-yeast invertase
secretion signal-AflII-GAL10 promoter/GAL1 promoter-AflII-chicken
PH-3-phosphoglycerate kinase gene terminator was added to the NotI
site in the polylinker. This new plasmid (pGET829) containing the
URA3 gene and dual expression units for chicken PDI and PH within
the disrupted TRP1 gene was cut with PmlI (61 bp from EcoRI in the
promoter region) and ApaLI (10 bp in from the PstI site on the other end
of the TRP1 gene) and therefore has homology to both ends of the TRP1
gene. Integration of this linear fragment was performed by a double
crossover during yeast transformation. Western assays using antibodies to chicken PH and PDI (32) identified the highest producing transformants. Subsequent analysis of the resulting strain, GY5382, indicated that it contains multiple integrations of the chicken PDI and PH
expression unit at the TRP1 locus.
Yeast Strains, Transformation, and CultureStrain GY5196 has the
genotype MATa leu21 trp163 ura3-52 his3200 GAL. Strain GY5382
(genotype MATa ura3-52 gal1102 trp1::(chicken PDI chicken PH
URA3)) contains repeated copies of chicken PH and PDI genes integrated into the TRP1 gene. GY5382 was selected from crosses with
other strains and screening for higher producing spores. After generating spheroplasts or using lithium treatment (33) with addition of 100 ng
of plasmid DNA, yeast transformants were selected on 2% agar plates
containing 2% dextrose, 0.67% yeast nitrogen base lacking amino acids
(Difco and Becton Dickinson Labware, Franklin Lakes, NJ), and 0.5%
casamino acids (and 24 g/ml uracil for GY5196 transformants) by
growing for 3 days at 30 C.
Each strain was grown in base medium consisting of yeast nitrogen
base buffered with 1% sodium citrate (pH 6.5) and supplemented with
a carbon source (20 g/liter galactose for GY5196 and 10 g/liter glucose
and 5 g/liter galactose for GY5382) and 0.5% casamino acids unless
otherwise described. Supplementation using arginine (110 mg/liter),
glutamate (765 mg/liter), and/or lysine (286 mg/liter) was at concentrations equivalent to the concentrations in casamino acids. Each culture
was grown at 20 C (without PH and PDI) and 30 C (with PH and
PDI) unless indicated otherwise and harvested at 60 70 h. The cells
were collected by centrifugation, resuspended in phosphate-buffered
saline plus 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride, mixed
with an equal volume of acid-washed glass beads, and frozen at 70 C.
The cells were thawed and lysed by vortexing for 6 15 min and centrifuged to remove cellular debris.
Quantitative Assay for CollagenCollagen yield was determined by
a luminometric immunoassay utilizing a goat anti-type I collagen antibody from BIODESIGN International (Kennebunkport, ME) derivatized with either biotin or ruthenium chelate. Samples were analyzed by
lysing cells as described above and centrifuging to remove cell debris.
The clarified supernatant samples from cell lysis were diluted in matrix
buffer (100 mM PIPES (pH 6.8) and 1% (w/v) bovine serum albumin) in
duplicate. A 25-l aliquot was mixed with 50 l of an antibody solution
containing 1 g/ml ruthenium chelate-conjugated antibody and
1.5 g/ml biotin-conjugated antibody in diluent (matrix buffer plus 1.5%
Tween 20). Samples were incubated for 2 h at 20 C with shaking. A
25-l aliquot of 1 mg/ml solution of streptavidin-conjugated magnetic
beads (in diluent) was added to each sample, and the samples were
shaken for 30 min. A 200-l aliquot of ORIGEN assay buffer (IGEN
Inc., Gaithersburg, MD) was added to each sample and then placed in
an ORIGEN analyzer (IGEN Inc.). Total protein was determined using
the BCA assay (Pierce) using a microtiter plate format.
Gels and Western BlotsThe equivalent of 20 ml of cells at A600 1.0
were collected, resuspended in 200 l of buffer, and lysed as described
above. SDS sample buffer was added; the samples were incubated at
100 C for 5 min; and the debris was collected by centrifugation. Clarified supernatants were loaded onto 5 or 10% SDS-polyacrylamide gels,

Type I Procollagen Expression in Yeast

electrophoresed, and stained using either GELCODE blue (Pierce) or
silver stain.
Protein was transferred from gels onto polyvinylidene difluoride
membranes (Millipore Corp., Bedford, MA) using Western blotting and
probed using an antibody against the N-propeptide region (LF-39,
1:15,000 dilution) or C-propeptide region (LF-41, 1:10,000 dilution) of
human 1(I) procollagen (34) and a horseradish peroxidase-linked secondary detection antibody (Pierce), followed by development using the
ECLTM detection kit (Amersham Pharmacia Biotech).
Carbohydrate AnalysisPlasmid pGET327 in strain GY5344, an
early strain containing the integrated PH and PDI expression cassette, was grown, and the cells were lysed as described above. Procollagen was precipitated from the clarified supernatant with 4.5 M NaCl
and resuspended in 0.1 M Tris-HCl (pH 7.4). Recombinant procollagen
C-proteinase/BMP-1 (35) was used to cleave at the C-propeptide junction (36). The digest was treated with endoglycosidase H (New England
Biolabs Inc., Beverly, MA) to remove N-linked carbohydrates as described by the manufacturer. The digests were analyzed by Western
blotting using the 1(I) procollagen C-propeptide-specific antibody
LF-41 (34).
Determination of Thermal StabilityPepsin digestions were performed on yeast extracts at pH 2.5 using 640 units/ml pepsin with
incubation for 15 min. The samples were neutralized with 1 M Tris base.
SDS sample buffer was added, and the samples were boiled and then
loaded onto a 5% SDS-polyacrylamide gel. Type I procollagen purified
from yeast cells or from conditioned medium of human skin fibroblasts
was treated with a mixture of trypsin (100 g/ml) and chymotrypsin
(250 g/ml) (37). The samples were preheated to the desired temperature for 15 min in a thermal cycler (Perkin-Elmer Model 480), followed
by addition of proteases and further incubation for 2 min. The digestion
was stopped by addition of SDS sample buffer, followed by immediate
boiling of the samples.
Amino Acid AnalysisAliquots of the purified protein samples were
dried and then subjected to vapor-phase hydrolysis overnight at 116 C
under N2 in vacuo. The hydrolyzed amino acids were derivatized with
the AccQ-Tag chemistry kit from Waters and analyzed on an AccQ-Tag
column using a Hewlett-Packard Model 1100 high pressure liquid chromatography apparatus.
Circular Dichroism AnalysisPurified samples were diluted to 100
g/ml using 200 mM sodium phosphate (pH 7.0). Aliquots of 200 l were
analyzed using a Jasco (Easton, MD) Model J-715 CD spectropolarimeter with a Peltier controlled sample holder. The samples were equilibrated for 5 min at each temperature and then scanned from 250 to
185 nm. The results were plotted as the molar ellipticity at a given
wavelength as a function of temperature. A first derivative plot of the
data was used to determine Tm.
RESULTS

Expression of 1(I) Procollagen Gene and Analysis of Collagen ProteinThe initial approach to producing human type I
procollagen was to express the 1(I) procollagen polypeptide.
The native human 1(I) procollagen signal sequence was tested
as well as the prepro-HSA (23) and prepro--factor (38) sequences that are commonly used to express heterologous proteins in yeast (Fig. 1). Yeast cells were transfected with plasmids that contain the 1(I) procollagen gene constructs varying
only in their signal sequence (Fig. 2A). The native procollagen
signal sequence resulted in the highest level of 1(I) procollagen produced based on the intensity of the bands on the Western blot; the prepro--factor and prepro-HSA regions also directed the synthesis of human procollagen, but to a lesser
degree. As expression of 1(I) procollagen in the absence of
2(I) procollagen results in homotrimer formation in mammalian cells (9), yeast extracts were treated with pepsin to digest
susceptible proteins. A light band was detected at the expected
size for collagen on SDS-polyacrylamide gel (Fig. 2B), indicating the presence of a homotrimeric collagen triple helix.
Since earlier reports described hyperglycosylation in several
yeast strains (39), we compared the sizes of N-linked oligosaccharide at the single acceptor site located in the C-propeptide of
1(I) procollagen (40). The C-propeptide trimer was removed
from procollagen with C-proteinase/BMP-1 (41). Following deglycosylation of human skin fibroblast- and yeast-derived hu-

23305

FIG. 1. Illustrations of the expression cassettes for type I procollagen. The locations of 1 and 2(I) procollagen, PH, and PDI
genes in the expression constructs are indicated by shaded rectangles.
Each promoter is shown as a box with an arrow to indicate the direction
of transcription; the GAL1/GAL10 promoter is bidirectional. The 1(I)
procollagen gene was expressed from the GAL10 side of the bidirectional promoter in all constructs. PH and PDI expression cassettes
were placed in the TRP1 gene when integrated into the yeast genome.
PGK, 3-phosphoglycerate kinase gene.

man procollagen C-propeptides with endoglycosidase H, identical decreases in molecular mass were seen by Western
blotting (Fig. 2C). This result suggests that similar levels of
carbohydrate were added to human procollagen expressed in
Saccharomyces and in fibroblast cultures.
The same signal sequence substitutions tested for 1(I) procollagen plus the yeast invertase signal were used for expression of the individual gene products 2(I) procollagen, PH,
and PDI. The native signal sequences for 2(I) procollagen and
PH gave the highest expression detected by Western blotting,
whereas PDI was more efficiently expressed and secreted into
the endoplasmic reticulum with the preinvertase signal sequence than with its native signal sequence. In addition, no
difference was measured in the expression levels or retention of
PDI in the endoplasmic reticulum by Western blotting when
the endoplasmic reticulum retention signal KDEL, present in
PDI of higher eucaryotes, was replaced with HDEL, present in
yeast PDI (data not shown).
The formation of a triple helix of homotrimeric type I procollagen stable at 25 C would indicate the functionality of the
prolyl hydroxylase enzyme. The Tm for non-hydroxylated procollagen is 25 C (3). We compared pepsin-digested extracts
from Saccharomyces expressing human 1(I) procollagen at
30 C with and without the genes that code for prolyl hydroxylase. One prominent band was seen on SDS-polyacrylamide
gel that comigrated with the expected size of 1(I) procollagen
in extracts from cells containing the PH and PDI genes, but
this band was absent from cells without prolyl hydroxylase
(Fig. 2D). In the absence of the hydroxylation system, the pro-1
chains were not able to fold into a triple helix that was stable at
the 30 C growth temperature. These data suggest the yeast cells
are expressing and assembling an active prolyl hydroxylase
tetramer, resulting in the formation of hydroxyproline, which
stabilizes the triple helix at elevated temperatures.
Expression of Heterotrimeric Type I ProcollagenThe next
step was to express four genes in Saccharomyces to test
whether they are sufficient for production of type I procollagen.
To accurately measure procollagen expression levels, an immunoassay was developed to quantify human heterotrimeric

23306

Type I Procollagen Expression in Yeast

TABLE I
Expression of type I procollagen by S. cerevisiae containing various
combinations of prolyl hydroxylase subunits
Plasmid

Type I
procollagen genes

PH/PDI

Yeast strain

h/ca
c/c
h/h
c/cb
c/cb

GY5196
GY5196
GY5196
GY5196
GY5382
GY5382

Expression level

g/mg protein

pGET150
pGET837
pGET901
pGET903
pGET150
pGET737

1/2
1/2
1/2
1/2

0
0.11
0.27
0.05
0
0.71

h denotes the human gene; c denotes the chicken gene.

b
Multiple PH/PDI expression units were integrated into the chromosome.

FIG. 2. Characterization of type I procollagen homotrimer. A,

Western blot of yeast extracts from strains expressing 1(I) procollagen
with different signal sequences. Transfected Saccharomyces cells were
grown at 20 C, and expression of the 1(I) procollagen gene was induced by galactose. Yeast extracts were subjected to electrophoresis and
Western blotting using antibody LF-39, which recognizes the N-propeptide of human 1(I) procollagen. Procollagen containing its native signal sequence is pGET327; that containing the prepro-HSA signal is
pGET323; and that containing the -factor signal is pGET335. B, pepsin digestions at different temperatures of yeast extracts expressing
1(I) procollagen (pGET327). Pepsin-digested yeast extracts were electrophoresed on SDS-polyacrylamide gel, and proteins were visualized
by silver staining. C, carbohydrate analysis of the homotrimeric type I
procollagen C-propeptide region expressed from plasmid pGET327. Endoglycosidase H (EndoH) was used to cleave N-linked oligosaccharide
from the C-propeptide of both the human skin fibroblast (HSF)- and
yeast-expressed human type I procollagen homotrimers. The digests
were reduced and electrophoresed on SDS-polyacrylamide gel, followed
by Western blotting. The C-propeptide was identified using the LF-41
antibody. D, pepsin digestions of the type I procollagen homotrimer
from pGET327 expressed in yeast with and without prolyl hydroxylase/
protein-disulfide isomerase genes integrated into the yeast. Pepsindigested yeast extracts were electrophoresed on SDS-polyacrylamide
gel, and proteins were visualized by silver staining.

type I collagen. This assay was challenged to detect thermally

denatured human placental type I collagen, pepsin-resistant
human type I collagen homotrimer expressed in our yeast
system, or bovine type I collagen. No signal was detected.
Several different expression units were generated that either
placed the genes on a 2 vector or integrated them into the
yeast genome (Fig. 1). Both procollagen genes were derived
from human sequences; the PH and PDI genes were either of
chicken or human origin.
Expression constructs that contained chicken/chicken PH
and PDI subunits expressed type I procollagen at higher levels
than human/human or human/chicken prolyl hydroxylase subunits (Table I). The integration of the chicken prolyl hydroxylase genes into the yeast genome further increased type I
procollagen expression. Other plasmid constructs tested but
not reported here included different combinations of yeast promoters driving the four genes. In addition, a new strain was

created by integrating the 1(I) and 2(I) procollagen genes

into the yeast chromosome. The results of these experiments
were lower or undetectable levels of procollagen expression
(data not shown).
Characterization of Recombinant Type I CollagenThe folding of type I procollagen into a heterotrimeric helix, the Tm of
the resulting helix, and the level of hydroxyproline in the
helical region were determined. The thermal stability of recombinant type I collagen was evaluated by treatment with a
mixture of trypsin and chymotrypsin at various temperatures
(37). The yeast-derived recombinant collagen heterotrimer
(Fig. 3A) was resistant to the proteases at temperatures as high
as 40 C. The melting curves for the fibroblast-derived collagen
(Fig. 3B) suggested that this collagen had a slightly higher Tm
relative to the recombinant collagen. Circular dichroism measurement of purified type I collagen showed a Tm of 35 C
(Fig. 4), which is slightly below the Tm of tissue-derived collagen. To directly demonstrate the presence of hydroxyproline in
the recombinant collagen, amino acid analysis was performed
on a purified sample. This analysis showed hydroxyproline
levels that were 82 2% (n 7) of values for collagen from
tissue-derived sources, which is in agreement with the CD
measurements. Direct detection of hydroxyproline residues by
amino acid analysis unequivocally demonstrated the functionality of the prolyl hydroxylase enzyme in our strains.
Medium OptimizationDuring development of the expression system, experiments had shown that 0 2% casamino acid
supplementation of the medium influenced the level of detectable human procollagen, with 0.5% casamino acids as the optimal concentration. Further analysis of the medium using
three additional supplementations at 0 1% was undertaken to
optimize procollagen production (Table II). Casamino acid supplementation was compared with the medium supplements
Bacto-Tryptone, Bacto-peptone, and yeast extract for their influence on procollagen expression. A level of 0.5% casamino
acid supplementation supported the highest levels of procollagen production of the different supplements tested.
Since casamino acids were the most stimulatory for procollagen production, simpler amino acid mixtures, based on the
concentrations that would be found in medium containing 0.5%
casamino acids, were tested to identify the stimulatory component(s). Several combinations of amino acid mixtures were
tried. Increased levels of proline and glycine had no effect on
procollagen production levels. Ultimately, yeast nitrogen base
supplemented with arginine, glutamate, and lysine or with
glutamate alone supplied the needed component necessary
for high level procollagen production (Table II). Procollagen
expression levels were 3 4 g/mg of total protein, or 0.3
0.4%. This requirement of hydroxylated procollagen for precursors of -ketoglutarate in the media suggests that not
enough -ketoglutarate is made in vivo for the hydroxylation
reaction. Ascorbic acid (another cofactor of prolyl hydroxy-

Type I Procollagen Expression in Yeast

23307

FIG. 3. Thermal stability of collagen expressed in S. cerevisiae.

Collagen purified from pGET737 in the GY5382 strain (A) or procollagen from human skin fibroblast-conditioned medium (B) was treated
with a mixture of trypsin and chymotrypsin at various temperatures.
Digests were electrophoresed on SDS-polyacrylamide gel, and proteins
were visualized by staining with GELCODE blue. Lane 1, human collagen purified from placenta; lane 2, undigested sample; lanes 310,
enzyme digests at 25, 30, 33, 35, 38, 40, 42, and 45 C, respectively.

lase) supplementation of the medium had no effect on hydroxylation and production of the heterotrimer.
DISCUSSION

The results of this study demonstrate that a complex, multisubunit procollagen molecule can be synthesized and assembled into a triple helix in Saccharomyces cerevisiae containing
a functional multisubunit prolyl hydroxylase enzyme. Three
procollagen chains, coded by two genes, are synthesized and
assembled into a triple helix. Two genes coding for prolyl hydroxylase form an active enzyme, presumably a tetramer.
Prolyl hydroxylase post-translationally modified procollagen,
resulting in a thermally stable molecule. Therefore, the posttranslational modification must occur in the same location
within the endoplasmic reticulum as the assembly of the three
procollagen chains. Saccharomyces apparently lacks only the
prolyl hydroxylase required for production of thermally stable
human type I procollagen. The prolyl hydroxylase stabilizes the
triple helix through the generation of hydroxyproline residues,
but also is associated with the folding of the triple helix. PDI
supports the folding and disulfide formation of procollagen
C-propeptides (7). The low levels of triple helix detected by
expressing the 1(I) procollagen gene in the absence of transfected prolyl hydroxylase genes suggest the existence of a less
efficient or rate-limited mechanism in Saccharomyces for the
generation of triple helical procollagen. It can be postulated
that the endogenous yeast PDI may assemble the three procollagen polypeptide chains, but the winding of the triple helix is
more efficient in the presence of prolyl hydroxylase.
The procollagen molecule produced in this expression system
had several features of tissue-derived procollagen synthesized
by mammalian cells. The procollagen molecule was triple helical and thermally stable with a Tm of 35 C based on CD
analysis. Non-hydroxylated collagen has a Tm of 2325 C,
whereas collagen isolated from mammalian tissues has a Tm of
39 40 C (3). The Tm was consistent with the stability of the
triple helix to proteolytic digestion and was in agreement with
the level of hydroxyproline determined by amino acid analysis
equivalent to 82% of levels found in tissue-derived type I collagen. N-Linked carbohydrate was also detected on recombi-

FIG. 4. Circular dichroism analysis of type I procollagen purified from S. cerevisiae. A, purified human type I collagen was equilibrated at each temperature and then scanned. The temperature measurements are indicated (). Normalized ellipticity was plotted against
temperature. The scale of 100 indicates triple helical collagen, and 0 is
denatured collagen. B, the Tm was determined as 35 C from the maximum of the first derivative of the curve in A.

nant type I procollagen. Since earlier reports describe hyperglycosylation of heterologous proteins in several yeast strains,
the size of N-linked oligosaccharide at the single acceptor site
located in the C-propeptide of 1(I) procollagen was determined. The C-propeptide contained carbohydrate with a mass
equivalent to that found in procollagen isolated from skin fibroblasts. No evidence of hyperglycosylation was observed for
the 1(I) procollagen polypeptide.
Both homotrimeric and heterotrimeric type I procollagens
were synthesized by Saccharomyces using plasmids that contained one or both procollagen genes, respectively. The procollagen genes and the genes for prolyl hydroxylase were sufficient to assemble procollagen polypeptides and to generate
thermally stable procollagen. To obtain and to optimize expression of procollagen, gene location, use of different promoters,

23308

Type I Procollagen Expression in Yeast

TABLE II
Expression of type I procollagen by S. cerevisiae using different
medium component additions
Procollagen production
Medium
Exp. 1

Exp. 2

g/mg total protein

YNBa 0.25% CAA

YNB 0.5% CAA
YNB 1% CAA
YNB 0.25% BT
YNB 0.5% BT
YNB 1% BT
YNB 0.25% BP
YNB 0.5% BP
YNB 1% BP
YNB 0.25% YE
YNB 0.5% YE
YNB 1% YE
YNB
YNB REK
YNB E

2.34
4.24
1.54
1.92
1.81
1.14
0.76
1.15
1.46
1.74
2.33
1.53
0.26

3.64

3.71
3.44

a
YNB, yeast nitrogen base; CAA, casamino acids; BT, Bacto-Tryptone; BP, Bacto-peptone; YE, yeast extract; REK, arginine, glutamate,
and lysine; E, glutamate.

signal sequence modifications, and species origin of the posttranslational machinery were varied. All of these parameters
played key roles in the production of thermally stable human
type I procollagen, with some combinations producing little or
no detectable procollagen material. The configuration that gave
the highest type I procollagen yield was placement of the human 1(I) and 2(I) procollagen genes on a 2 vector with
integration of the two chicken prolyl hydroxylase genes into the
yeast genome. We initially tested chicken PH and PDI, but
expected human PH and PDI to offer equal or better posttranslational modification efficiency and to potentially increase
procollagen levels indirectly through increased folding and procollagen thermal stability. Instead, higher expression of human
procollagen was measured with chicken prolyl hydroxylase
genes (PH and PDI) compared with their human counterparts. Two possibilities to explain the higher expression of
human procollagen using chicken prolyl hydroxylase are potentially higher enzymatic activity and enhanced interaction of
the gene with its promoter to increase transcription. Sequence
homologies between chicken and human PH protein and
cDNA sequences are 88 and 81%, respectively (28). Chicken
and human PDI protein sequences are 90% homologous,
whereas cDNA sequence comparison shows only 76% homology
(26).
Developmental work on this yeast expression system showed
the requirement of casamino acids for higher level production
of recombinant procollagen. Addition of individual components
of casamino acids to the medium showed that glutamate alone
was sufficient to provide procollagen expression levels equivalent to those observed using the entire mixture of amino acids
found in casamino acids. One possible explanation for the role
of glutamate is its ability to undergo intracellular oxidation to
create -ketoglutarate, an intermediate in the tricarboxylic
acid cycle and an essential cofactor for proline hydroxylation.
Glycine and proline supplementation did not affect procollagen
expression levels even though procollagen consists of high
amounts of these two amino acids. In addition, this medium
does not contain ascorbic acid, a cofactor for prolyl hydroxylase
and necessary for the expression of thermally stable procollagens in other recombinant expression systems (14, 18). Saccharomyces must generate sufficient levels of this cofactor to supply to the prolyl hydroxylase enzyme. These results are
important for the production of proteins that could be used in
medical applications, as use of a medium free of animal-derived

components provides an extra margin of safety and avoids a

potential regulatory hurdle defining the source of a raw
material.
Fibroblasts have been used to delineate the biosynthetic
pathway of collagen for 3 decades. Since fibroblasts express
procollagen ubiquitously, many suggestions have been made
about the function of various proteins in the biosynthesis of
procollagen at the level of transcription, translation, and assembly. Hsp47 is a serpin-like collagen-specific chaperone localized in the endoplasmic reticulum that transiently binds to
type IV collagens and that is involved in the assembly and/or
packaging of collagens (8). Hsp47 is associated with polysomebound 1(I) procollagen chains (42), and it prevents overmodification of type III procollagen in transfected 293 kidney cells
(43). BiP/Grp78 and Grp94 form a complex with Hsp47 during
the maturation of newly synthesized type IV collagen (44). PDI
interacts with the C-propeptide of collagen chains prior to
trimer formation, and prolyl hydroxylase remains associated
with the triple helical domain if triple helical formation is
prevented (7). Yeast was chosen as a model system for the
synthesis of procollagen because it is a well characterized eucaryotic organism that does not normally synthesize procollagen. To synthesize type I procollagen, only four genes were
shown to be required. This study shows that other fibroblastspecific genes are not needed for the basic mechanism of recognition and assembly of the procollagen polypeptides to form
a stable triple helical molecule. Recombinant Hsp47 was not
required for assembly of triple helical type I procollagen. PDI
expressed with PH increased homotrimeric type I procollagen
synthesis, suggesting an enhancement of the assembly of procollagen with increased PDI and/or PH protein.
In summary, we have demonstrated the minimum requirements for type I procollagen expression in Saccharomyces. A
total of four genes are required. Two genes code for the two
polypeptide chains of human type I procollagen. Two additional
genes code for prolyl hydroxylase, a modification enzyme that
post-translationally hydroxylates proline residues within the
triple helical domain of the procollagen polypeptides. Prolyl
hydroxylase or its individual subunits also enhance the level of
procollagen synthesized. Glutamate, possibly acting as a precursor for the synthesis of -ketoglutarate, is required for generating high levels of triple helical procollagen molecules.
Other proteins associated with procollagen synthesis in mammalian cells are not required in Saccharomyces. It remains to
be determined if specific chaperone proteins play a subtle role
in the assembly of procollagens in eucaryotic cells.
AcknowledgmentsWe thank Arcie Alea, Meghan Bowzer, Hein Bui,
Betty Elder, Lucas Hanscom, Nathan C. Hitzeman, Doug Hodges,
Caroline Lanigan, Chin Y. Loh, Paul Nguyen, Winson Ong, Naomi
Sakai, and Ernie Tai for excellent technical work on this project. We
also thank Rudolf Jaenisch and Daniel Greenspan for 1 and 2(I)
procollagen cDNAs, Winson Kao for chicken PH and PDI cDNA clones,
and Larry Fisher for LF-39 and LF-41 antibodies.
REFERENCES
1. Haralson, M. A., and Hassell, J. R. (1995) in Extracellular Matrix: A Practical
Approach (Haralson, M. A., and Hassell, J. R., eds) pp. 130, IRL Press,
New York
2. Prockop, D. J., and Kivirikko, K. I. (1984) N. Engl. J. Med. 3111, 376 386
3. Berg, R. A., and Prockop, D. J. (1973) Biochem. Biophys. Res. Commun. 52,
115120
4. Privalov, P. L. (1982) Adv. Protein Chem. 35, 53104
5. McLaughlin, S. H., and Bulleid, N. J. (1998) Matrix Biol. 16, 369 377
6. Fleischmajer, R., Olsen, B. R., Timpl, R., Perlish, J. S., and Lovelance, O.
(1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3354 3358
7. Wilson, R., Lees, J. F., and Bulleid, N. J. (1998) J. Biol. Chem. 273, 96379643
8. Nagata, K. (1996) Trends Biochem. Sci. 21, 2226
9. Geddis, A. E., and Prockop D. J. (1993) Matrix 13, 399 405
10. Fertala A., Sieron, A. L., Ganguly, A., Li, S. W., Ala-Kokko, L., Anumula, K. R.,
and Prockop, D. J. (1994) Biochem. J. 298, 3137
11. Fichard, A., Tillet, E., Delacoux, F., Garrone, R., and Ruggiero, F. (1997)
J. Biol. Chem. 272, 3008330087
12. Vuori, K., Pihlajaniemi, T., Marttila, M., and Kivirikko, K. I. (1992) Proc. Natl.

Type I Procollagen Expression in Yeast

Acad. Sci. U. S. A. 89, 74677470
13. Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. (Tokyo) 121,
10611069
14. Myllyharju, H., Lamberg, A., Notbohm, H., Fietzek, P. P., Pihlajaniemi, T., and
Kivirikko, K. I. (1997) J. Biol. Chem. 272, 21824 21830
15. Lamberg, A., Helaakoski, T., Myllyharju, J., Peltonen, S., Notbohm, H.,
Pihlajaniemi, T., and Kivirikko, K. I. (1996) J. Biol. Chem. 271,
11988 11995
16. Toman, P. D., Pieper, F., Sakai, N., Karatzas, C., Platenburg, E., de Wit, I.,
Samuel, C., Dekker, A., Daniels, G. A., Berg, R. A., and Platenburg, G.
(1999) Transgenic Res. 8, 415 427
17. John, D. C. A., Watson, R., Kind, A. J., Scott, A. R., Kadler, K. E., and Bulleid,
N. J. (1999) Nature Biotechnol. 17, 385389
18. Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., and Kivirikko, K. I.
(1997) EMBO J. 16, 6702 6712
19. Vaughan, P. R., Galanis, M., Richards, K. M., Tebb, T. A., Ramshaw, J. A. M.,
and Werkmeister, J. A. (1998) DNA Cell Biol. 17, 511518
20. Chen, C., Oppermann, H., and Hitzeman, R. (1984) Nucleic Acids Res. 12,
8951 8970
21. Johnston, M., and Davis, R. (1984) Mol. Cell. Biol. 4, 1440 1448
22. Stacey, A., Mulligan, R., and Jaenisch, R. (1987) J. Virol. 61, 2549 2554
23. Hitzeman, R. A., Chen, C. Y., Dowbenko, D. J., Renz, M. E., Liu, C., Pai, R.,
Simpson, N. J., Kohr, W. J., Singh, A., Chisholm, V., Hamilton, R., and
Chang, C. N. (1990) Methods Enzymol. 185, 421 440
24. Kurjan, J., and Herskowitz, I. (1982) Cell 30, 933943
25. Lee, S.-T., Smith, B. D., and Greenspan, D. S. (1988) J. Biol. Chem. 263,
13414 13418
26. Kao, W. W.-Y., Nakazawa, M., Aida, T., Everson, W. V., Kao, C. W.-C., Seyer,
J. M., and Hughes, S. H. (1988) Connect. Tissue Res. 18, 157174
27. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllyla, R., Huhtala, M.-L.,
Koivu, J., and Kivirikko, K. I. (1987) EMBO J. 6, 643 649

23309

28. Bassuk, J. A., Kao, W. W.-Y., Herzer, P., Kedersha, N. L., Seyer, J., DeMartino,
J. A., Daugherty, B. L., Mark, G. E., III, and Berg, R. A. (1989) Proc. Natl.
Acad. Sci. U. S. A. 86, 73827386
29. Helaakoski, T., Vuori, K., Myllyla, R., Kivirikko, K. I., and Pihlajaniemi, T.
(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4392 4396
30. Stinchcomb, D. T., Struhl, K., and Davis, R. W. (1979) Nature 282, 39 43
31. Fasiolo, F., Bonnet, J., and Lacroute, F. (1981) J. Biol. Chem. 256, 2324 2328
32. Berg, R. A., Kao, K. K., and Kedersha, N. L. (1980) Biochem. J. 189, 491 499
33. Becker, D. M., and Lundblad, V. (1996) Current Protocols in Molecular Biology,
Vol. 2, pp. 13.7.113.7.4, John Wiley & Sons, Inc., New York
34. Fisher, L. W., Lindner, W., Young, M. F., and Termine, J. F. (1989) Connective
Tissue Res. 21, 4350
35. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Driz,
R. W., Hewick, R. M., and Wang, E. A. (1988) Science 292, 1528 1534
36. Hojima, Y., van der Rest, M., and Prockop, D. J. (1985) J. Biol. Chem. 260,
15996 16003
37. Bruckner, P., and Prockop, D. J. (1981) Anal. Biochem. 110, 360 368
38. Zsebo, K. M., Lu, H.-S., Fieschko, J. C., Goldstein, L., Davis, J., Duker, K.,
Suggs, S. V., Lai, P.-H., and Bitter, G. A. (1986) J. Biol. Chem. 261,
5858 5865
39. De Baetselier-Van Broekhoven, A. (1994) Bioprocess Technol. 19, 431 447
40. Clark, C. C. (1979) J. Biol. Chem. 254, 10798 10802
41. Kessler, E., Adar, R., Goldberg, B., and Niece, R. (1986) Collagen Relat. Res. 6,
249 266
42. Sauk, J. J., Smith, T., Norris, K., and Ferreira, L. (1994) J. Biol. Chem. 269,
39413946
43. Hosokawa, N., Hohenadl, C., Satoh, M., Kuhn, K., and Nagata, K. (1998)
J. Biochem. (Tokyo) 124, 654 662
44. Ferreira, L. R., Norris, K., Smith, T., Hetert, C., and Sauk, J. J. (1996) Connect.
Tissue Res. 33, 265273