The document discusses research into producing recombinant forms of the Cynara cardunculus Cyprosins, which are aspartic proteinases with milk clotting activity found in cardoon flowers. Researchers constructed expression cassettes containing genes for the Cyprosins with different signal sequences and expressed them in Saccharomyces cerevisiae. Cyprosin A was produced intracellularly but was inactive. Cyprosin B was actively secreted when expressed with the MFα1 presequence and native prosequence. A site-directed mutagenesis was performed to change the active site sequence of Cyprosin B from Asp-Ser-Gly to Asp-Thr-Gly as in other aspartic proteinases, but

4. Summary
The flowers of Cynara cardunculus (cardoon) produce at least two types of
glycosylated aspartic proteinases with proteolytic and milk clotting activity, the Cyprosins
and Cardosins. This flowers have been used for centuries in Portugal in the home-scale
production of high quality cheep-milk cheese. However, the industrial utilisation of this new
source of clotting enzymes is limited by the variability and uncontrolled factors inherent to
the biological system. Production of the enzymes via recombinant DNA techniques offers
the possibility to obtain large amounts of purified proteins suitable for industrial
applications. The heterologous production of glycosylated proteins demands the use of an
eukaryotic host capable of conducting the post-translation mechanisms. Saccharomyces
cerevisiae is the genetic and biochemical most well characterised organism for heterologous
protein production. It has a secretory system similar to the higher eukaryotic cells, and even
secrets a small portion of its own proteins which facilitates the recovery and purification of
the desired protein. Moreover S. cerevisiae has no pathogenic relationship to humans, it
lacks detectable endotoxins and is generally recognised as a safe organism for the production
of food and medical products.
The aspartic proteinases share a similar three-dimensional structure and active site,
and are generally thought to be synthesised as inactive preproforms known as zymogens,
which are then converted to the active mature forms by proteolytic removal of first, the N-
terminal presequences and after the N-terminal prosequence. Removal of the presequence is
performed in the endoplasmic reticulum by the signal peptidase. In most cases acidification
triggers the removal of the prosequence and processing of the enzyme to the mature active
form. The prosequence is consider to mask the active-site region intramolecularly, keeping
the propeptide inactive at higher pH. The plant aspartic proteinases have additionally
another intramolecular region, the plant-specific-insert (PSI), which is though to be removed
during propeptide processing. This region is considered to attach the proprotein to the
membranes during its transit through the secretory system, being removed upon delivery to
its final destination. Moreover, it forms a loop which masks the active-site region preventing
unwanted activation. Once it is removed, the prosequence occupies the active-site region
and controls the proprotein activation.
The aim of this work was to develop a system to obtain recombinant forms A and B
of the Cynara cardunculus Cyprosins. We have initiated this purpose to obtain recombinant
Cyprosins by the determination of the signal peptidase cleavage site of both enzymes,
expressed respectively by the genes CYPRO1 and CYPRO11 (Chapter 2). Subsequently,
we have constructed several expression cassettes by using either the native preprosequence
of both genes, the presequence of the yeast SUC2 gene, the presequence of the yeast MF1
gene and the preprosequence of this last gene. The various gene constructs were cloned
under the control of the GAL7 promotor and the PGK1 terminator (Chapter 2). These
expression cassettes were then transferred to an E. coli - yeast shuttle low copy plasmid
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5. based on the YCplac111 (Gietz and Sugino, 1988) and/or to a 2 multicopy vector based on
the YEplac181 (Gietz and Sugino, 1988). Different strains of Saccharomyces cerevisiae
were used as hosts: BJ1991 (MAT leu2 trp1 ura3-52 prb1-1122 pep4-3), BJ2168 (MATa
leu2 trp1 ura3-52 prc1-407 prb1-1122 pep4-3), MT302/1c=a (arg5-6 leu2-12 his3-11 his3-
15 pep4-3 ade1) and W303-1A (MAT leu2-3,112 ura 3-1 trp1-1 his3-11,15 ade2-1 can1-
100 GAL SUC2). The results concerning gene expression were analysed by quantification of
the steady-state mRNA levels using Northern blots. They indicate that the transcription
levels of the gene-constructs in all combinations of vector type and yeast strain used is not
the limiting factor, and therefore, not responsible for possible differences in the production
of Cyprosins by the yeast cells (Chapter 2).
Cyprosin A was only produced by the yeast cells using homologous signal
sequences, although it accumulated intracellularly and was degraded at the late exponential
growth phase (Chapter 3). The recombinant protein showed a higher apparent molecular
mass than expected and had neither proteolytic nor milk clotting activity. In all cases tested
the protein produced by the yeast cells was not secreted into the culture medium (Chapter
3). The lack of secretion and the inactivity of the intracellularly accumulated
protein, followed by degradation, was proposed to be due to misfolding of the expressed
Cyprosin A, probably because the post-translational processing mechanisms in this
heterologous system are not adequate.
Production of a fully active Cyprosin B and secretion into the culture medium was
better achieved by combining the presequence of MF1 with the sequence coding the
proprotein (Chapter 4). The use of the entire leader sequence of MF1 resulted in
intracellular accumulation of heterogeneous intermediates with high apparent molecular
mass. It is possible that they represent hyperglycosylated intermediates and unprocessed
forms of the protein due to overloading the Ste13p and/or Kex2p, the enzymes responsible
for removing the MF1 prosequence. The presequence of the yeast MF was found to be
sufficient to drive secretion of Cyprosin B, and the native prosequence was necessary for the
production of an active 32.5 kDa protein (Chapter 4). The use of low-copy-number CEN-
derived plasmids gave better results than the multicopy 2-derived plasmids, either
related to the intracellular accumulated or secreted protein yields. From all strains
tested the protease deficient BJ1991 strain was the best in producing and secreting an active
protein, showing high proteolytic and milk clotting activity (Chapter 4). Secretion of the
Cyprosin B was dependent on the BJ1991 growth phase. Maximum activities were obtained
at the stationary growth phase in YPGal medium (A600 nm=10), while in middle exponential
phase (A600=2), the yeast cells secreted an inactive 47.5 kDa protein, thought to be an
unprocessed form, in which the plant-specific-insert, characteristic of all aspartic
proteinases, had not yet been removed. The studies performed using the Endo-H enzyme
showed that the secreted
32.5 kDa protein corresponding to the large subunit of the yeast Cyprosin B was
glycosylated with a high mannose type glycan chain at its unique putative N- glycosylation
site (Chapter 4).
A common aspect of all aspartic proteinases is the presence of two aspartate
residues at the active site which are part of a consensus sequence Asp-Thr/Ser-Gly. The
30

6. mammalian, microbial and virus aspartic proteinases have two conserved active site
triads
Asp-Thr-Gly. Some fungi aspartic proteinases and all plant enzymes are exceptions to this
consensus sequence. There second active site triad contains a serine residue instead of
threonine (Asp-Ser-Gly). In the present state of our knowledge the physiological relevance
of this change is unknown. Neither we know whether this change is responsible for
differences reported in the milk clotting and proteolytic activity when these enzymes are
compared with enzymes from other origin, such as Chymosin. However, a few authors
showed that those differences are responsible for different organoleptic properties of the
cheese prepared with bovine milk using these two classes of clotting enzyme. In order to
clarify this question, we have used a site-directed-mutagenesis protocol and we have
changed the serine residue at the Asp290-Ser-Gly (DSG) active site of Cyprosin B by the
threonine residue (Chapter 5). Comparison of the proteolytic and milk clotting activity of
the heterologous mutated and non-mutated protein, produced by Saccharomyces cerevisiae
BJ1991 strain, showed that there are no significant differences concerning these parameters.
We conclude that substitution of the serine residue on the second active site of plant aspartic
proteinases is not the reason for the differences observed on the proteolytic activity of the
proteins produced by Cynara cardunculus and eventually by the other plants when compared
to the aspartic proteinases from other origin (Chapter 5).
Plant aspartic proteinases share another common aspect which is not observed in its
animal or microbial counterparts. This is the presence of an intramolecular insert of
about
104 amino acids known as the plant-specific-insert. This insert appears to be removed during
the protein maturation giving rise to the dimeric form characteristic of these enzymes. It is
also been implicated in the inactivation of the zymogen and acquisition of the proper protein
folding during the processing steps before zymogen activation. The alignment of ten plant
aspartic proteinases shows that the PSI sequences contain low homology, although a few
motifs and conserved amino acids can be observed (Chapter 6). With the aim of studying
the relevance of PSI on the aspartic proteinases activity, we have deleted the PSI from the
CYPRO11 gene, coding for Cynara cardunculus Cyprosin B. The results of SDS-PAGE
show that the curtailed protein, which is still inside the cell, has a molecular mass higher
than the native protein. The secreted protein containing the deletion was hardly detected by
the CCMP1 antiserum, and also had a molecular mass higher, than the native protein. The
proteolytic activity of the secreted PSI-deleted-Cyprosin decreased one third as compared to
the non-deleted Cyprosin. While the secreted Cyprosin showed an high milk clotting activity
the PSI-deleted-Cyprosin lost this activity. It was shown that the lack of PSI in the nascent
protein affects both the proteolytic and the milk clotting activity of Cyprosin. Moreover,
during protein proper processing PSI has to be removed in order to form a fully active
enzyme (Chapter 6).
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7. Chapter 5
the original plasmid (Fig 2). Figure 3 shows the resulting pattern analysed in a 1% agarose
gel. In this figure the undigested profile of the pYCC11preMF plasmid is shown in lane UC.
pUC19 plasmid was included in the restriction reaction as a control for the enzyme activity.
Digestion with KpnI opened the plasmid which showed the expected 2.89 kb band (white
arrow in figure 3). Lanes 1, 2 and 3 show the pYCC11preMFDSG plasmid DNA isolated from
three E. coli clones, after digestion with KpnI enzyme. The resulting band obtained from
linearization of the plasmid (8284 bp) shows that this restriction site was introduced into the
plasmid pYCC11preMF, and so was the codon mutation. This result was further confirmed
by sequence analysis.
... GGT TGT GCA GCA ATT GCC GAC TCT GGA ACC TCT TTG TTG GCA GGT CCA ACG...
…CCA ACA CGT CGT TAA CGG CTG AGA CCT TGG AGA AAC AAC CGT CCA GGT TCG ...
... GGT TGT GCA GCA ATT GCC GAC ACA GGT ACC TCT TTG TTG GCA GGT CCA ACG...
... CCA ACA CGT CGT TAA CGG CTG TGT CCA TGG AGA AAC AAC CGT CCA GGT TCG...
Fig. 2 - Details of the cloning strategy used to replace the serine residue at the DSG active site by the threonine
residue. In the original sequence the codon for serine (TCT) was replaced by the codon for threonine (ACA).
This was done by designing specific primers containing the KpnI restriction site, introduced by silent mutation,
and with which PCR fragments were created. Digestion of the two PCR fragments created respectively by using
the DSG1 primer and the DSG2 primer, introduced the desired mutation and the restriction site into the mutated
sequence.
114
Original sequence
BsgI
G C A A I A D S G T S L L A G P T
BsgI KpnI HindIII
… GGT TGT GCA GCA ATT GCC GAC ACA GGT AC C AAG CTT GCG GC …
… CCA ACA CGT CGT TAA CGG CTG TGT C CA TGG TTC GAA CGC CG ... DSG1
BamHI KpnI
DSG2 … C CGC GGA TCC GGT AC C TCT TTG TTG GCA GGT CCA ACG…
… G GCG CCT AGG C CA TGG AGA GGC AAC CGT CCA GGA TCG...
Ligation
Mutated sequence
BsgI KpnI
G C A A I A D T G T S L L A G P T

8. The effects of replacing the serine residue by threonine in the Cyprosin DSG active site
The effects of the serine substitution
The aspartic proteinases are a highly homologous class of proteases which have two
conserved active sites containing aspartate residues. Their homology is particularly high
within the region around and at the active sites. Most aspartic proteinases from plants,
animals and fungi show motifs at the two active site regions, namely VIFDTGSSNLWVPS
and AIVDTGTSLL (Fig. 4). The aspartic proteinases from plant origin are an exception.
Although they contain approximately the same consensus sequences around the active sites,
the threonine residue of the second active site was replaced by the serine residue. This
exception seems to be specific for the plant enzymes and for the yeast Saccharomycopsis
fibuligera and Candida albicans aspartic proteinases (Fig. 4). The biological meaning of this
substitution is not known.
Fig. 3 - Agarose gel (1%) showing three E. coli clones containing the introduced KpnI
restriction in the mutated gene (Ser  Thr). The absence of the KpnI restriction site in
the original plasmid sequence, pYCC11preMF, is shown in the second lane by the uncut
(UC) plasmid pattern. The pUC19 2.686 Kb plasmid containing a unique KpnI site, was
set into the restriction reaction as a control for the enzyme activity (white arrow). Lanes
1, 2 and 3 show the band for the pYCC11preMFDSG open plasmid (8.033 Kb) . M - 1 Kb
Plus DNA Ladder Marker (Gibko BRL, Pasley, UK). Black arrows show the DNA
markers 12, 8 and 2 Kb, respectively from top to bottom.
The active site of the Cyprosins, the aspartic proteinases from the dried flowers of
Cynara cardunculus, includes the two aspartic acids residues Asp100, Asp290 for the Cyprosin
A and Asp103, Asp287
for the Cyprosin B (Brodelius et al, 1998), as determined from the
putative amino acid primary structure. For both enzymes the second active site is the
one
containing the serine substitution. Whether this substitution is the basis of differences in the
activity of the plant enzymes as compared with the enzymes from other origin or whether it
has an effect on the specificity of these enzymes for the substrate, is not known. A
comparison of the caseinolytic specificity of the aspartic proteinases obtained from
flowers
115
M UC 1 2 3

9. Chapter 5
B
Fig. 5 - Analysis of the effects of replacing the serine residue at the DSG active site by the threonine residue. A
- Western blot showing intracellular Cyprosin production (left) and secretion into the YPGal culture medium
(right), by S. cerevisiae BJ1991 strain transformed with the original plasmid, pYCC11preMF (lane 1) which
was set as control, and the mutated plasmid pYCC11preMFDSG (lane 2). P- proteins from a plant crude extract.
Arrows show the position of the 32.5 and 31 MW marker. B - Proteolytic activity determined as the percentage
of relative activity per total secreted protein in the samples described in A. C - Milk clotting activity analysis
using the culture medium from the samples described in A.
120
%RF/ugProtein
Intracellular Extracellular
A P 1 2 P 1 2
1 2
C
14
12
10
8
6
4
2
0
1 2