APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2588–2595 Vol. 70, No.

5
0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.5.2588–2595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its

Expression in Microbial Cells
Rosario Muñoz,1 José L. Garcı́a,2 Alfonso V. Carrascosa,1 and Ramon Gonzalez1*
Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC),1 and Department of Molecular
Microbiology, Centro de Investigaciones Biológicas (CSIC),2 Madrid, Spain
Received 22 October 2003/Accepted 15 January 2004

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main
protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this
enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive
precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for
bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag
libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen.
The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences,
which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced
functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is
activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme
generates digestion profiles with ␣-, ␤-, or ␬-casein indistinguishable from those obtained with a natural
pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioac-
tive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that
there is no risk of transmission of bovine spongiform encephalopathy.

Bovine pepsin is a member of the pepsin-like family of
aspartic peptidases found in the fourth stomach of cows. Pep-
tidases belonging to this family exhibit optimal activity at an
acidic pH and contain two active-site aspartate residues re-
quired for function. Like other gastric proteinases, bovine pep-
sin is synthesized as an inactive precursor or zymogen, pep-
sinogen. Pepsinogen contains a prosegment at the N-terminal
end, which prevents access of the substrate to the active site
and maintains the enzyme in its inactive form. The conversion
of pepsinogen to active pepsin is initiated by acidic conditions
and involves several conformational changes and bond cleav-
age steps that result in proteolytic removal of the prosegment
and release of the active site (20).
The main industrial application of bovine pepsin is in cheese
making; this enzyme is either naturally present in calf stomachs
or is added as a less expensive complement in calf chymosin
preparations (2, 6). The fluctuations in the availability and
price of calf rennet some years ago stimulated the search for
alternative milk coagulants; this search included cloning and
expression in several microorganisms of a cDNA coding for
calf prochymosin (3, 17, 28). Recombinant bovine chymosin
was first commercialized in 1990 (5); sales of this compound
quickly accounted for more than one-half of the market for
rennet, and several versions, produced from genetically engi-
neered Escherichia coli, Kluyveromyces lactis, or Aspergillus ni-
ger, became available. The recombinant rennets have been
shown to be equivalent to natural rennet (18). After the bovine
spongiform encephalopathy crisis in Europe, food safety con-
* Corresponding author. Mailing address: Department of Microbi-
ology, Instituto de Fermentaciones Industriales (CSIC), Juan de la
Cierva 3, 28006 Madrid, Spain. Phone: 34-915622900, ext. 359. Fax:
34-915644853. E-mail: rgonzalez@ifi.csic.es.
siderations also supported the use of recombinant rennet in-
stead of natural rennet. Whereas milk safety has been clearly
documented by both epidemiological and experimental data
(http://www.tseandfoodsafety.org/position_papers/position
_paper_on_the_safety_o/tafs_position_paper_on_the_2.pdf), the
safety of the abomasum from ruminants is inferred based partially
on a number of assumptions, such as the quality of feed and the
procurement of the abomasum (http://www.emea.eu.int/pdfs
/human/bwp/033702en.pdf). Indeed, prions have been detected
(although no infectivity has been titrated) in the abomasum of
experimentally infected small ruminants (http://europa.eu.int
/comm/food/fs/sc/ssc/out296_en.pdf).
Its limited proteolytic activity makes chymosin the best milk-
clotting enzyme in terms of curd yield, and in addition, the
off-flavors often associated with excess proteolysis are ab-
sent. However, ripening of cheese requires additional pro-
teolytic activities, which come in part from cheese micro-
biota, including the starter cultures used for acidification
and in part from the residual proteolytic activity of the
enzymes used for milk clotting. The main changes attribut-
able to proteolysis during cheese ripening are changes in the
texture and flavor. The effect on flavor is due to the release
of peptides and amino acids, which, in turn, can be the
substrates for further reactions resulting in aroma com-
pounds, such as deamination, decarboxylation, or desulfu-
ration (7). The acceptable degree of proteolysis during rip-
ening depends on the type of cheese, and several strategies
have been proposed for accelerating this process in order to
reduce production costs. These strategies include the use of
elevated temperatures (13), addition of proteolytic enzymes
(13), and addition of bacteriocin-producing starters (8, 15,
16). It has been suggested that the different enzyme speci-
ficities of bovine pepsin and chymosin may contribute favor-

2588
VOL. 70, 2004
TABLE 1. Sequences of the primers used in this study for PCR amplification or for site-directed mutagenesis
Primer
cDNA-PB3 ...................................5⬘-CCCGGGCTGCAGAATTCATGTCTGTTGTCAAGATCCCACTCGTCAAAAAGAAGTCC-3⬘
cDNA-PB2 ...................................5⬘-GTGGCGGCCTCCCGGATG-3⬘
Xba-PB.........................................5⬘-GCTCTAGAGGGTATTAATAATGAGCGTCGTCAAAATCCCACTCG-3⬘
Hind-PB .......................................5⬘-GCGAAGCTTAGTTAGCTATTAGGCCACGGGAGCCAGGCCG-3⬘
BP05A ..........................................5⬘-GGAATATTAAGCTTGGTACCGAGCTCGAATTCATGAGATTTCCTTCAATTTTTACTGC-3⬘
BP05B...........................................5⬘-GATCTTGACAACAGACATAGCTTCAGCCTCTCTTTTATCC-3⬘
PT7-PB .........................................5⬘-GGTTTCCCTCTAGAAATAATTTGTTTAACTTTAAGAAGGAGATATACATATGAGCGTCGTCAA-3⬘
ably to the quality of cheeses made with natural rennet
preparations containing pepsin (30).
Several pepsin-encoding genes, including the genes from
pigs (26) and chickens (21), have been cloned, and some of
them have been expressed in microorganisms (26); however,
remarkably, the gene coding for bovine pepsin has not been
completely described so far. Only the N-terminal amino acid
sequence of bovine pepsinogen (10) and a partial genomic
sequence erroneously labeled as the sequence encoding bovine
pepsinogen have been determined previously (14). In this pa-
per we describe cloning of a complete cDNA encoding bovine
pepsinogen and expression of this cDNA in bacterial and yeast
cells. The use of the technology described in this work is cov-
ered by Spanish patent application 200300179.
MATERIALS AND METHODS
Materials. cDNA clones from two different bovine cDNA libraries were used
as starting material for construction of a complete cDNA coding for bovine
pepsinogen. Clones 1Abo01C03, 1Abo04B07, 1Abo09A06, 1Abo10A02,
1Abo11A02, 1Abo15A08, 1Abo15G06, and 1Abo16A03 (GenBank accession
numbers BG937426, BG937636, BG937863, BG937943, BG938081, BG938289,
BG938334, and BG938347, respectively) were obtained from a bovine abomasum
cDNA library (1Abo) in the Uni-ZAP XR vector (Stratagene, La Jolla, Calif.)
and were kindly provided by S. Moore and C. Hansen from the University of
Alberta, Edmonton, Alberta, Canada. Clones MARC3BOV 85L15,
MARC3BOV 103I10, MARC3BOV 103G10, and MARC3BOV 105H9 (Gen-
Bank accession numbers BF774958, BM106242, BM106232, and BM106659,
respectively) were obtained from a mixed-tissue bovine cDNA library
(MARC3BOV) in pCMV-SPORT6 (Invitrogen, Leek, The Netherlands) and
were obtained from the Children’s Hospital Oakland Research Institute. Purified
milk proteins and other chemicals were purchased from Sigma-Aldrich (St.
Louis, Mo.). Stabo 230, a commercial enzyme solution containing approximately
85% bovine pepsin and 15% chymosin, was kindly provided by Christian Hansen
A/S (Hoersholm, Denmark).
Microbial strains, plasmids, and growth conditions. Escherichia coli DH5␣F⬘
[F⬘ endA1 hsdR17 (rk⫺mk⫹) supE44 thi-1 recA1 gyrA(Nalr) relA1 ⌬(lacIZYA-
argF)U169 deoR (␾80dlac⌬(lacZ)M15; Promega] was used for all DNA manip-
ulations, as well as for pepsinogen production. E. coli JM109(DE3) [endA1 recA1
gyrA96 hsdR17 supE44 relA1 thi ⌬(lac-pro) F⬘ (traD36 proAB⫹ lacIq lacZ⌬M15)
␭cI857 ind1 Sam/nin5 lacUV5-T7 gene 1; Promega] and Saccharomyces cerevisiae
BY4741 (MATa his3-⌬1 leu2-⌬0 met15-⌬0 ura3-⌬0) were used for expression of
recombinant bovine pepsinogen. pIN-III(lppp-5)A3 and pT7-7, two expression
vectors carrying isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promot-
ers, were used for expression in E. coli strains (11, 25). pYES2, an episomal
expression vector for S. cerevisiae carrying the GAL1 galactose-inducible pro-
moter, was used for expression in yeast.
E. coli strains were cultured in Luria-Bertani (LB) medium (22) at 37°C and
200 rpm. When required, ampicillin was added to the medium at a concentration
of 100 ␮g ml⫺1. Yeast cells were cultured in YPD medium (1% yeast extract, 2%
peptone, 2% glucose) at 30°C.
Recombinant DNA techniques. Restriction endonucleases and T4 DNA ligase
were obtained from Roche (Basel, Switzerland) and were used according to the
recommendations of the supplier. Gel electrophoresis analyses of plasmids, re-
striction fragments, and PCR products were performed in agarose gels as de-
scribed previously (22). Bacterial plasmids were purified by the alkaline sodium
dodecyl sulfate (SDS) method (22). Yeast chromosomal DNA was purified by
RECOMBINANT BOVINE PEPSINOGEN PRODUCTION 2589
Sequence
the method of Querol et al. (19). In vivo excision of phagemids from
LambdaZAP clones was performed as suggested by Stratagene. Insertion of PCR
fragments into preexisting constructs was performed by using a QuikChange kit
from Stratagene, as described by Wang and Malcolm (29). PCR amplification
was performed by using Pfu DNA polymerase (Stratagene) and the instructions
of the supplier. The sequences of the primers used in this work are shown in
Table 1.
DNA sequencing was carried out with an ABI Prism 377 DNA sequencer
(Applied Biosystems, Inc., Branchburg, N.J.). Sequence similarity searches were
carried out by using the Basic Local Alignment Search Tool (BLAST) (1) with
the EMBL and GenBank databases.
Transformation of microorganisms. Transformation of E. coli was performed
by electroporation (22), and the transformants were selected on LB medium
supplemented with ampicillin (100 ␮g ml⫺1). Transformation of S. cerevisiae was
performed by the method described by Gietz and Woods (9). Yeast transfor-
mants were selected on SD medium without uridine or uracil (0.67% yeast
nitrogen base [Difco, Detroit, Mich.], 2% glucose, 60 mM leucine).
Induction and preparation of cell extracts. Bovine pepsinogen cDNA was
expressed in E. coli as follows. Bacterial cells harboring either the original vector
or the recombinant plasmid were grown in LB broth supplemented with ampi-
cillin (100 ␮g ml⫺1) at 37°C and 200 rpm to an optical density at 600 nm of 0.6.
The cultures were subsequently shifted to 20 or 37°C, and synthesis of bovine
pepsinogen was induced by adding 0.5 mM (final concentration) IPTG. At
different times, samples of the cultures were harvested by centrifugation (10,000
⫻ g, 5 min), and the bacterial cells were recovered in 50 mM Tris-phosphate
buffer (pH 7.0) and disrupted by sonication. The insoluble fractions were sepa-
rated by centrifugation (15,000 ⫻ g, 15 min), and the supernatants were used for
enzyme assays.
Expression in S. cerevisiae was induced with galactose as follows. Yeast cells
harboring either pYES2 or pBP05 were grown on SD medium at 30°C and 150
rpm until the mid-exponential phase and then transferred to induction medium.
Depending on the experiment, a culture containing 2 ⫻ 107 cells per ml was
transferred to SG minimal induction medium (0.67% yeast nitrogen base [Difco],
2% galactose, 60 mM leucine) or to YPG rich induction medium (1% yeast
extract, 2% peptone, 2% galactose) and incubated under the same conditions.
Samples were withdrawn at different times, the cells were separated by centrif-
ugation (12,000 ⫻ g, 5 min), and the supernatants were assayed for proteolytic
activity. In order to obtain the cell extracts, about 108 cells were harvested by
centrifugation at 10,000 ⫻ g for 5 min and resuspended in 500 ␮l of 50 mM
Tris-phosphate buffer (pH 7). The cell suspension was vortexed at full speed for
5 min in the presence of 0.5 g of glass beads. Insoluble material was removed by
centrifugation at 12,000 ⫻ g for 5 min, and the supernatants were used to
measure cell-associated proteolytic activity.
Renaturation of inclusion bodies. Extracts of E. coli JM109(DE3) cells har-
boring plasmid pBP06 induced overnight at 37°C with IPTG were used as the
starting material for renaturation of the inclusion bodies. Purification and rena-
turation of the inclusion bodies were performed as described previously for
recombinant bovine chymosin expressed in E. coli (4).
Milk-clotting assays. Supernatants of galactose-induced S. cerevisiae cultures
were dialyzed and freeze-dried. Pepsinogen was activated to pepsin as previously
described (12). Briefly, 0.3 M HCl was added to the solution until the pH was 2.0,
and after incubation for 10 min at room temperature the pH was raised to 6.0 by
adding a cold solution of 4 M sodium acetate. Changes in the pH of the solution
were continuously monitored with a pH meter. Milk-clotting activity was assayed
in microtiter plates as described by Emtage et al. (4). Each well contained 100 ␮l
of 12% (wt/vol) dried skim milk, 20 mM CaCl2, 25 mM sodium phosphate buffer
(pH 6.3), and an appropriate dilution of the commercial or activated recombi-
nant enzyme. The plates were incubated at 37°C and after 30 min were inverted
to allow nonclotted milk to drain. Clotting activity was confirmed by the presence
2590 MUÑOZ ET AL. APPL. ENVIRON. MICROBIOL.

of a white coagulate at the bottom of the well and was recorded by scanning the
plates against a black background with a Scanjet 5470c scanner (Hewlett-Pack-
ard, Camas, Wash.).
Protease assays. The amount of functional pepsinogen in a cell extract or in
the culture medium was estimated by the method of Kasell and Meitner (12) by
using bovine hemoglobin (Sigma-Aldrich) as the substrate. Briefly, 350 ␮l of
hemoglobin substrate prepared as described by Kasell and Meitner (12) was
incubated with 350 ␮l of enzyme diluted in 0.01 M HCl–0.1 M NaCl (pH 2.0) at
37°C for 30 or 60 min. The reaction was stopped by adding 700 ␮l of 5%
trichloroacetic acid, the mixture was incubated for 15 min on ice and centrifuged
at 15,000 ⫻ g for 15 min, and the supernatant was used to determine the optical
density at 280 nm. Activities were expressed as equivalents of porcine pepsin
(Sigma-Aldrich) assayed under the same conditions. The amount of active pepsin
after pepsinogen activation (see above) was estimated as follows. First, 125 ␮l of
azocasein substrate (2% azocasein in 50 mM sodium phosphate buffer [pH 6.0])
was incubated with 75 ␮l of enzyme diluted in 50 mM sodium phosphate buffer
(pH 6.0). The reaction was stopped by incubation with 0.6 ml of 10% trichloro-
acetic acid for 15 min at 4°C, the reaction mixture was centrifuged at 12,000 ⫻
g for 15 min, and 0.6 ml of the supernatant was mixed with 0.7 ml of 1 M NaOH
before the optical density at 440 nm was recorded. Isolated milk proteins were
digested as described by Ustunol and Zeckzer (27), with minor modifications.
Briefly, ␣-, ␤-, and ␬-caseins (Sigma-Aldrich) were dissolved in 0.1 M phosphate
buffer (pH 6.7) at a final concentration of 2 mg ml⫺1. Each standardized protease
solution was diluted 10-fold in the same buffer and allowed to react overnight at
30°C. The hydrolysis profiles were visualized by SDS—15% polyacrylamide gel
electrophoresis (PAGE).
Nucleotide sequence accession number. The sequence of the reconstituted
cDNA determined in this study has been deposited in GenBank database under
accession number AY330769.

RESULTS
Cloning of a complete version of bovine pepsinogen cDNA.
Sequences corresponding to human, camel, and pig pepsin-
encoding genes, as well as to bovine chymosin-encoding genes,
were used to perform a BLAST search of sequences potentially
coding for bovine pepsin. In this way two cDNA libraries con-
taining partial bovine pepsin cDNA sequences were identified
in the expressed sequence tag database. Sequence analysis of
the 1Abo and MARC3BOV clones showed that they contained
the 3⬘ end of the cDNA, including the poly(A) tail. Compari-
son of the hypothetical sequence of the cDNA clones with the
sequence described previously for the first 110 amino acids of
pepsinogen (10) revealed that the clones from the
MARC3BOV library had a 118-bp internal deletion. However,
clones from the 1Abo library did not have any internal dele-
tion. Clones MARC3BOV 103I10 and 1Abo04B07 were used
to obtain a complete version of bovine pepsinogen cDNA.
Figure 1 shows an alignment of the N-terminal sequence of
pepsinogen with the sequences encoded by both cDNA.
The strategy used to construct a complete version of the
bovine gene coding for pepsinogen is summarized in Fig. 2. A
DNA fragment containing the sequence coding for bovine
pepsinogen that is missing in clone 1Abo04B07 was generated
by PCR performed with primers cDNA-PB2 and cDNA-pB3
by using MARC3BOV 103I10 as the template. Primer cDNA-
PB3 contains the ATG start codon and the sequence coding for
the four N-terminal amino acids of pepsinogen. This PCR
fragment was then inserted into 1Abo04B07 by using a
QuikChange kit from Stratagene, as described in Materials and
Methods. The resulting plasmid, pBP01, contained a cDNA
sequence coding for a complete version of bovine pepsinogen
in the phagemid vector pBluescript SK(⫺).
Sequence analysis. The peptide sequence of bovine pepsino-
gen is 55% identical to the calf prochymosin sequence and 83
FIG. 1. Sequence alignment showing the experimentally deter-
mined N-terminal sequence of bovine pepsinogen (BovPgn) (accession
number P00792), the hypothetical translated sequences of cDNA
clones 1Abo04B07 (04B07) (accession number BG937636) and
MARC3BOV 103I10 (103I10) (accession number BM106242), the
sequence of rabbit pepsinogen III (RabPgn) (accession number
AAA85370), and the sequence previously assigned to bovine pepsino-
gen exons 6, 7, and 8 (P111) (accession number JT0398). The nucle-
otides that are different in the bovine pepsinogen exon 6, 7, and 8 and
rabbit pepsinogen III sequences and the bovine pepsinogen cDNA
clone sequence are underlined in the bovine pepsinogen exon 6, 7, and
8 sequence. The conserved positions for the introns in the genomic
sequence for most mammalian aspartic protease genes are indicated by
vertical lines. The sequence downstream of the 118-bp internal dele-
tion in MARC3BOV 103I10 has been translated in a different frame.
The sequences of both cDNA clones were experimentally determined
in this work because the sequence in the GenBank database is a partial
sequence.
and 82% identical to the pig and human pepsinogen se-
quences, respectively. The levels of similarity are 94% for pig
pepsinogen, 91% for human pepsinogen, and 71% for calf
prochymosin. These values are very similar to those obtained
with the corresponding mature peptides. Two copies of the
consensus sequence for eukaryotic aspartic proteases, flanking
the aspartate residues of the catalytic site as described in
PROSITE entry PS00141 (24), are present in the deduced
sequence of bovine pepsinogen at positions 75 to 86 and 258 to
269. Remarkably, although the N-terminal sequence of the
cDNA is identical to the known sequences of pepsinogen
genes, there are several differences between the previously
VOL. 70, 2004 RECOMBINANT BOVINE PEPSINOGEN PRODUCTION 2591

FIG. 2. Construction of plasmid pBP01 containing a complete version of bovine pepsinogen cDNA. The 118-bp internal deletion on
MARC3BOV 103I10 is indicated by a dashed line. The positions of primers cDNA-PB2 and cDNA-PB3 are indicated by arrows. Sequences arising
from the insert in MARC3BOV 103I10 are indicated by solid arrows, and sequences arising from the insert in 1Abo04B07 are indicated by open
arrows. Vector sequences are represented by a single line [pBluescript SK(⫺)] or a double line (pCMV-SPORT6). pgnA, bovine pepsinogen cDNA
sequences; E, EcoRI; H, HindIII; N, NcoI; P, PstI; S, SalI; Sc, ScaI; X, XhoI. (Sc) indicates a ScaI site absent in the MARC3BOV 103I10 clone
due to the 118-bp internal deletion.

published partial cDNA sequence of exons 6, 7, and 8 of the
bovine pepsinogen gene (14) and the cDNA sequence de-
scribed here (Fig. 1) (the overall genomic structures of most
mammalian aspartic proteases are similar, with eight introns at
conserved positions). Two years ago we used the partial pep-
sinogen sequence described by Lu et al. (14) to amplify bovine
pepsinogen gene sequences from calf genomic DNA (unpub-
lished data). To our surprise, the amplified DNA sequences
(accession number AY442187) clearly differed from the se-
quences described previously. In fact, the coding region of this
fragment is identical to the pepsinogen cDNA elucidated in
this work (data not shown). In order to solve this puzzle, we
performed further sequence comparisons of these three exon
sequences with the whole nucleotide databases using BLAST.
Surprisingly, this analysis revealed 100% identity between the
sequence of the three exons and the sequence of the rabbit
pepsin gene, suggesting that the exons actually are rabbit pep-
sinogen exons and not bovine pepsinogen exons as described
previously (Fig. 1).
Expression of bovine pepsinogen in E. coli. To express bo-
vine pepsinogen in E. coli, the cDNA coding for pepsinogen
was PCR amplified from pBP01 by using Pfu DNA polymerase
and primers Xba-PB and Hind-PB. Primer Xba-PB is based on
the sequence of plasmid pIN-III(lppp-5)A3 and contains the
ribosome binding site domain, as well as the initial start codon
and the sequence encoding the first five amino acid residues of
pepsinogen, which were designed by preferred codon usage by
E. coli. Primer Hind-PB is also based on the sequence of
pIN-III(lppp-5)A3 and contains the sequence encoding the six
last amino acid residues of pepsinogen and several stop codons
arranged in tandem (Table 1). The purified PCR fragment was
digested with XbaI and HindIII, whose target sequences were
incorporated into the primers and cloned into pIN-III(lppp-
5)A3 digested with the same restriction enzymes. The resulting
plasmid, pBP03, contained bovine pepsinogen cDNA under
control of the lppp-5 and lacpo promoters, which can be in-
duced at high levels by IPTG (11). The sequence and the site
of insertion of the cDNA were verified by restriction analysis
and DNA sequencing. Cell extracts were prepared from E. coli
DH5␣ cells harboring recombinant plasmid pBP03. To detect
the recombinant protein, cell extracts were analyzed by SDS-
PAGE (Fig. 3A). Control cells containing the pIN-III(lppp-
5)A3 vector plasmid alone did not show expression over the
4-h time course analyzed, whereas expression of an additional
40-kDa protein was apparent with E. coli DH5␣ cells harboring
pBP03. The molecular mass of this protein was in good agree-
ment with the molecular mass deduced from the nucleotide
sequence of the bovine pepsinogen cDNA (40 kDa).
Cell extracts were also assayed for protease activity against
bovine hemoglobin at low pH. Cell extracts of E. coli DH5␣
harboring recombinant plasmid pBP03 exhibited protease ac-
tivity ranging from 0.2 ␮g (noninduced conditions) to 0.5 ␮g
(induced conditions) of porcine pepsin equivalents per ml of
culture, whereas no activity was detected in cell extracts pre-
pared from control cells containing the vector plasmid.
To improve the production of recombinant bovine pepsino-
gen, we transferred the bovine pepsinogen cDNA to the ex-
pression vector pT7-7. By using the strategy described above,
an XbaI/HindIII-digested PCR fragment containing bovine
pepsinogen cDNA was cloned in pT7-7 digested with the same
restriction enzymes. The resulting plasmid, pBP02, was used as
a template for PCR amplification with Pfu DNA polymerase
2592 MUÑOZ ET AL.
FIG. 3. SDS-PAGE analysis of soluble cell extracts of IPTG-in-
duced cultures of E. coli JM109(DE3) bearing recombinant plasmids.
(A) Expression on pIN-III(lppp-5)A3. Lane 1, E. coli DH5␣/pIN-
III(lppp-5)A3; lane 2, E. coli DH5␣/pBP03. (B) Expression on pT7-7.
Lane 1, E. coli JM109(DE3)/pT7-7; lane 2, E. coli JM109(DE3)/
pBP06. The arrows indicate the position of the overproduced protein.
The 12% polyacrylamide gels were stained with Coomassie blue. The
positions of molecular mass markers (SDS-PAGE standards; Bio-Rad)
are indicated on the left.
and primer PT7-PB. This primer provides the original ribo-
some binding site from pT7-7. The amplification product was
digested with DpnI to degrade the DNA from the original
plasmid, pBP02, and the amplified product was transformed
into the expression strain E. coli JM109(DE3). The resulting
plasmid, pBP06, contained bovine pepsinogen cDNA under
control of the T7 RNA polymerase-inducible ␾10 promoter.
Several conditions were tested to induce bovine pepsinogen
synthesis by E. coli JM109(DE3) cells harboring recombinant
plasmid pBP06. The values obtained for pepsinogen activity
were 2 orders of magnitude greater than the values obtained
for pBP03-transformed cells under similar culture conditions
(Table 2). Overnight (16-h) induction at 20°C resulted in the
maximum functional pepsinogen production. A similar conclu-
sion was drawn from an SDS-PAGE analysis. As shown in Fig.
3B, induction at 20°C for 16 h gave rise to a conspicuous
40-kDa protein band. We found that most, if not all, of the
protein was recovered from the supernatant of the cell extracts
TABLE 2. Proteolytic activities in cell extracts of E. coli
JM109(DE3) transformed with pBP06 under different IPTG
induction conditions
Proteolytic activitiesa
Conditions 20°C
4h 16 h 4h
Noninduced 0.7 ⫾ 0.2 0.5 ⫾ 0.2 ⬍0.3
Induced 4.5 ⫾ 1.3 43.0 ⫾ 7.0 ⬍0.3
a
The activities are expressed in milligrams of porcine pepsin equivalents per
gram (dry weight). The data are means ⫾ standard deviations for three separate
experiments.
APPL. ENVIRON. MICROBIOL.
TABLE 3. Proteolytic activities in the supernatants of S. cerevisiae
strains transformed with different plasmids
Proteolytic activitiesa
Medium
pYES2 pBP04 pBP05
SD ⬍0.2 ⬍0.2 ⬍0.2
SG ⬍0.2 ⬍0.2 0.9 ⫾ 0.3
YPD ⬍0.2 ⬍0.2 0.3 ⫾ 0.1
YPG ⬍0.2 ⬍0.2 1.9 ⫾ 0.5
a
The activities after 72 h of induction in different media (SD, SG, YPD, and
YPG media) are expressed in milligrams of porcine pepsin equivalents per gram
(dry weight). The data are means ⫾ standard deviations for three separate
experiments.
(Fig. 3B, lane 2), indicating that it was in a soluble form. The
40-kDa protein was absent in the supernatants prepared from
either an uninduced bacterial culture (Fig. 3B, lane 1) or E. coli
cells harboring only the vector plasmid (data not shown). We
observed a faint band of the same size with cell extracts from
cultures induced for 4 h at 20 and 37°C (data not shown). In
the case of the culture induced overnight at 37°C, we observed
an apparent 40-kDa band in the total cell extract, indicating
that the protein was in an insoluble form, probably inclusion
bodies, and this could explain the lack of proteolytic activity.
The formation of inclusion bodies was verified by direct ob-
servation of the induced cultures by phase-contrast micros-
copy. These bodies were observed in cultures induced over-
night at both 20 and 37°C; large amounts of functional
pepsinogen were recovered in cultures induced at 20°C,
whereas no functional protein was detected in cultures induced
at 37°C. Several unsuccessful attempts were made to recover
the functional protein from the inclusion bodies, based on the
procedure described for recombinant bovine chymosin (4).
Expression of bovine pepsinogen in S. cerevisiae. To produce
recombinant bovine pepsinogen in S. cerevisiae, a SacI/XhoI
fragment from pBP01 containing pepsinogen cDNA was
cloned into pYES2, a yeast episomic expression vector, which
was digested with the same enzymes. The resulting plasmid,
pBP04, contained the bovine pepsinogen cDNA under control
of the yeast GAL1 gene promoter, which is induced by galac-
tose and is repressed by glucose. When this system was used,
we were unable to detect any proteolytic activity in either the
culture broth or cell extracts from pBP04-containing yeast cells
that were induced by galactose in SG medium.
A different approach was used to promote secretion of pep-
sinogen by construction of a transcriptional fusion of pepsino-
gen cDNA with the alpha-factor secretion signal of S. cerevi-
siae. To do this, the sequence encoding the alpha-factor
secretion signal was PCR amplified from S. cerevisiae BY4741
genomic DNA by using primers BP05A and BP05B. The re-
sulting PCR product was used to introduce the secretion signal
into pBP04, which was fused upstream of the sequence coding
for bovine pepsinogen, with a QuikChange kit from Strat-
37°C
agene, as described in Materials and Methods. The resulting
16 h plasmid, pBP05, and pBP04 were introduced into S. cerevisiae
⬍0.3
BY4741 by transformation. The transformants were tested for
⬍0.3 pepsinogen production and secretion under different induction
conditions (Table 3). Only yeast strains harboring plasmid
pBP05 secreted pepsinogen into the culture medium. As ex-
pected, the amount of pepsinogen secreted was larger under
VOL. 70, 2004
FIG. 4. Time course of extracellular pepsinogen formation in yeast
cultures transferred to YPG induction medium. Symbols: F, pYES2-
transformed cells; ■, pBP05-transformed cells.
galactose induction conditions (SG or YPG medium) than
under glucose repression conditions (SD or YPD medium).
However, the yield was clearly increased by using complex
medium (YPG medium). A very low level of pepsinogen pro-
duction was detected in cell extracts, and only induced cultures
containing cells carrying plasmid pBP05 had intracellular pep-
sinogen levels slightly greater than the background level (data
not shown). There were no significant differences in intracel-
lular pepsinogen levels between cells carrying pYES2 and cells
carrying pBP04. The time course of induction on YPG medium
for cells carrying pBP05 was also investigated (Fig. 4); most of
the pepsinogen was released after between 6 and 24 h of
induction, but the levels continue to increase until 72 h.
Clotting activity. The clotting activity of recombinant bovine
pepsinogen produced in S. cerevisiae was determined as de-
scribed by Emtage et al. (4). Twofold dilutions of either re-
combinant acid-activated pepsinogen or Stabo 230 were added
to consecutive wells starting with an azocasein hydrolytic ac-
tivity equivalent to 4 ␮g of commercial porcine pepsin. Figure
5 shows the results of this experiment, which indicated that the
clotting activity of recombinant pepsin is similar to that of
Stabo 230.
Proteolytic activity against isolated milk proteins. We in-
vestigated the activities of native pepsin and S. cerevisiae-pro-
duced recombinant pepsin with purified cow milk caseins and
compared the activities under similar conditions. We studied
the extent and profile of hydrolysis of purified ␣-, ␤-, and
␬-caseins (Sigma-Aldrich) by using Stabo 230 or acid-activated
recombinant pepsinogen solutions. The enzyme solutions were
previously adjusted to contain similar proteolytic activities
against azocasein. Figure 6 shows the peptide profiles of ca-
seins after overnight incubation at 30°C in the presence of
Stabo 230 and in the presence of recombinant pepsin secreted
by S. cerevisiae. Both proteases showed the same peptide pat-
tern for the three substrates used, and although the extent of
FIG. 5. Milk-clotting activity of native pepsinogen (Stabo 230) or
recombinant pepsinogen obtained from yeast strains carrying plasmid
pBP05. The amount of enzyme used in each column is expressed in
microgram equivalents of porcine pepsin.
RECOMBINANT BOVINE PEPSINOGEN PRODUCTION 2593
hydrolysis with the recombinant protein was less than that with
Stabo 230 (Fig. 6B), this may have been due to a small differ-
ence in the activity units used for the assay.
DISCUSSION
Based on the results described above, we concluded that the
complete cDNA sequence encoding true bovine pepsinogen
was cloned and determined for the first time. This conclusion
was based on (i) the identity between the deduced protein
sequence and the known N-terminal sequence of bovine pep-
sinogen A, (ii) the high level of similarity between this se-
quence and that of pepsinogens from other mammalian spe-
cies, (iii) the size of the recombinant protein expressed in E.
coli, (iv) the proteolytic and milk-clotting activities of the re-
combinant protein, and (v) the comparison of casein digestion
profiles with the profiles produced by natural bovine pepsin. In
spite of the puzzling noticeable differences between the par-
tially described sequences of bovine pepsinogen exons 6, 7, and
8 and the cDNA sequence described in this paper, we demon-
strated that the previously described sequences exhibit 100%
identity with the rabbit pepsinogen gene, suggesting that they
were most likely obtained from a contaminated cDNA library.
It should be stressed that Lu et al. (14) failed to clone the
bovine chymosin gene from their library, even when they used
the homologous probe. In our case, we eliminated contamina-
tion since the cDNA fragments described here were derived
from two different bovine cDNA libraries and are identical in
the shared regions. Moreover, the hypothetical sequence trans-
lated from the cDNA sequence is identical to the first 110
amino acids of pepsinogen, and the coding sequence is iden-
tical to the coding sequence of the bovine genomic DNA frag-
ment previously cloned by us (data not shown) (accession num-
ber AY442187).
Recombinant bovine pepsinogen has been successfully pro-
duced in E. coli and S. cerevisiae. In E. coli the best expression
results were obtained by using strain JM109(DE3) as the host,
pT7-7 as the expression vector, and overnight induction with
IPTG at 20°C. Under these optimal conditions, pepsinogen
production in cell extracts, expressed in porcine pepsin equiv-
alents, was more than 40 mg/g (dry weight). The highest levels
of yeast-expressed pepsinogen were obtained by using a tran-
scriptional fusion of bovine pepsinogen cDNA with the S.
cerevisiae alpha-factor secretion signal and a complex medium
with galactose as the only carbon source (YPG medium) for
induction. Even though the yeast expression levels were lower
than those in E. coli, the use of pBP05 has the advantage that
the recombinant pepsinogen is recovered from the culture
broth. The secretion of the molecule facilitates both purifica-
tion and production in a continuous culture system.
The lack of pepsinogen production in yeast carrying plasmid
pBP04 may have been due to transcriptional problems. In this
plasmid the distance between the transcription start site and
the start codon is greater than the distance in pBP05; this is
due to the presence of pBluescript multiple-cloning-site se-
quences that have not been removed. Nevertheless, we cannot
exclude the possibility that secretion is necessary to avoid deg-
radation by intracellular yeast enzymes.
All attempts to recover functional pepsinogen from E. coli
inclusion bodies by using the strategies designed for recombi-
2594 MUÑOZ ET AL.
FIG. 6. SDS-PAGE analysis of isolated cow milk caseins treated with Stabo 230 or recombinant bovine pepsin (15% polyacrylamide gels).
(A) Proteolytic activity with ␣-casein; (B) proteolytic activity with ␤-casein; (C) proteolytic activity with ␬-casein. Lane 1, control; lane 2, Stabo
digestion; lane 3, soluble extract of S. cerevisiae carrying pBP05. The gels were stained with Coomassie blue. The positions of molecular mass
markers (SDS-PAGE standards; Bio-Rad) are indicated on the left.
nant bovine chymosin (4) have been unsuccessful. However, if
the large amounts of protein obtained as inclusion bodies in
cultures induced overnight at 37°C are taken into account,
fine-tuning of solubilization and refolding protocols for recom-
binant bovine pepsinogen is a potential target for improving
yield and purity.
One of the main potential applications of the recombinant
enzyme is to accelerate ripening in cured cheese or to produce
cheeses with properties similar to those made with rennet,
which naturally contains both chymosin and pepsin. Indeed, it
has been shown that ripening of Grana cheese is accelerated or
improved by using mixtures containing 10% bovine pepsin
compared to the ripening of cheese made by using recombi-
nant chymosin alone (30). Remarkably, successful cheese pro-
duction has been reported when 97% pure pepsin preparations
were used (2). To learn more about this possibility, we com-
pared the yeast-expressed recombinant bovine pepsinogen (af-
ter acid-induced activation) with a commercially available pep-
sin-rich preparation from bovine abomasum. The ratios of milk
clotting to general proteolytic activity, as measured with he-
moglobin or azocasein, were similar for the two enzymes.
When purified ␣-, ␤-, and ␬-caseins were used, the results were
also similar in terms of the degree of proteolysis and the main
hydrolysis products. Only in the case of ␤-casein was there a
small difference in the degree of hydrolysis, and this was prob-
ably due to slightly different amounts of enzyme used for the
assay. Fox and Wallace (7) showed that pepsin can hydrolyze
␤-casein at pH values near 2. However, degradation of ␤-ca-
sein by pepsin is dependent not only on pH but also on the
incubation temperature, and the protein is degraded more
rapidly at 2°C than at 32°C. It has been suggested that pepsin
is capable of degrading both ␣- and ␬-caseins but is unable to
degrade ␤-casein under the same experimental conditions (27).
However, under our experimental conditions ␤-casein was de-
graded by both the native bovine pepsinogen and the recom-
APPL. ENVIRON. MICROBIOL.
binant bovine pepsinogen, which generated the same peptide
profile.
In conclusion, this is the first time that a recombinant bovine
pepsinogen has been synthesized, and our findings pave the
way for using this enzyme as an alternative in cheese making;
use of this recombinant enzyme has the advantage that the
recombinant pepsin should be free from potential pathogenic
agents arising from animal tissues, particularly the causative
agent of bovine spongiform encephalopathy. In addition, like
recombinant bovine chymosin, recombinant pepsin would be
more acceptable than the native enzyme to vegetarian consum-
ers or to people subject to food restrictions due to religious
beliefs. Finally, other potential applications of bovine pepsin
include the release of bioactive peptides from casein mac-
ropeptide or caseins (23) or use as a general-purpose protease.
ACKNOWLEDGMENTS
We are grateful to C. Hansen and S. Moore for kindly providing
cDNA clones from the 1Abo library, to E. Cebollero for help with
yeast transformation, to F. Jorganes and A. Fernandez for technical
assistance, and to M. Sheehan for correcting the English version.
REFERENCES
1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:3389–3402.
2. Birkkjaer, H., and P. Jonk. 1985. Technological suitability of calf rennet
substitutes. Int. Dairy Fed. Bull. 194:8–13.
3. Cullen, D., G. L. Gray, L. J. Wilson, K. J. Hayenga, M. H. Lamsa, M. W. Rey,
S. Norton, and R. M. Berka. 1987. Controlled expression and secretion of
bovine chymosin in Aspergillus nidulans. Bio/Technology 5:369–376.
4. Emtage, J. S., S. Angal, M. T. Doel, T. J. R. Harris, B. Jenkins, G. Lilley, and
P. A. Lowe. 1983. Synthesis of calf prochymosin (prorennin) in Escherichia
coli. Proc. Natl. Acad. Sci. USA 80:3671–3675.
5. Flamm, E. L. 1991. How FDA approved chymosin: a case history. Bio/
Technology 9:349–351.
6. Foltmann, B. 1987. General and molecular aspects of rennets, p. 33–61. In
P. F. Fox (ed.) Cheese: chemistry, physics and microbiology, vol. 1. General
aspects. Elsevier Applied Science, London, United Kingdom.
7. Fox, P. F., and J. M. Wallace. 1997. Formation of flavor compounds in
cheese. Adv. Appl. Microbiol. 45:17–85.
VOL. 70, 2004
8. Garde, S., P. Gaya, M. Medina, and M. Nuñez. 1997. Acceleration of flavour
formation in cheese by a bacteriocin-producing adjunct lactic culture. Bio-
technol. Lett. 19:1011–1014.
9. Gietz, R. D., and R. A. Woods. 2002. Transformation of yeast by the LiAc/SS
carrier DNA/PEG method. Methods Enzymol. 350:87–96.
10. Harboe, M. K., and B. Foltmann. 1975. Bovine pepsin: the sequence of the
first 65 amino acid residues (completing the sequence of the first 110 resi-
dues of bovine pepsinogen). FEBS Lett. 60:133–136.
11. Inouye, S., and M. Inouye. 1985. Up-promoter mutations in the lpp gene of
Escherichia coli. Nucleic Acids Res. 13:3101–3110.
12. Kasell, B., and P. A. Meitner. 1970. Bovine pepsinogen and pepsin. Methods
Enzymol. 19:337–347.
13. Law, B. A. 2001. Controlled and accelerated cheese ripening: the research
base for new technologies. Int. Dairy J. 11:383–398.
14. Lu, Q., K. H. Wolfe, and D. J. McConell. 1988. Molecular cloning of multiple
aspartyl protease genes. Gene 71:135–146.
15. Martinez-Cuesta., M. C., P. Fernandez de Palencia, T. Requena, and C.
Pelaez,. 1998. Enhancement of proteolysis by a Lactococcus lactis bacteriocin
producer in a cheese model system. J. Agric. Food Chem. 46:3863–3867.
16. Morgan, S., R. P. Ross, and C. Hill. 1997. Increasing starter cell lysis in
Cheddar cheese using a bacteriocin-producing adjunct. J. Dairy Sci. 80:1–10.
17. Nishimori, K., Y. Kawaguchi, M. Hidaka, T. Uozumi, and T. Beppu. 1982.
Expression of cloned calf prochymosin genes sequence in Escherichia coli.
Gene 19:337–344.
18. Ortiz de Apodaca, M. J., L. Amigo, and M. Ramos. 1994. Study of the
milk-clotting and proteolytic activity of calf rennet, fermentation-produced
chymosin, vegetable and microbial coagulants. Milchwissenschaft 49:13–16.
19. Querol, A., E. Barrrio, and D. Ramón. 1992. A comparative study of different
methods of yeast strain characterisation. Syst. Appl. Microbiol. 15:439–446.
20. Richter, C., T. Tanaka, and R. Y. Yada. 1998. Mechanism of activation of the
gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin. Bio-
chem. J. 335:481–490.
RECOMBINANT BOVINE PEPSINOGEN PRODUCTION 2595
21. Sakamoto, N., H. Saiga, and S. Yasugi. 1998. Analysis of temporal expres-
sion pattern and cis-regulatory sequences of chicken pepsinogen A and C.
Biochem. Biophys. Res. Commun. 250:420–424.
22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
23. Shammet, K. M., R. J. Brown, and D. J. McMahon. 1992. Proteolytic activity
of proteinases on macropeptide isolated from ␬-casein. J. Dairy Sci. 75:1380–
1388.
24. Sigrist, C. J., L. Cerutti, N. Hulo, A. Gattiker, L. Falquet, M. Pagni, A.
Bairoch, and P. Bucher. 2002. PROSITE: a documented database using
patterns and profiles as motif descriptors. Brief Bioinform. 3:265–274.
25. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymer-
ase/promoter system for controlled exclusive expression of specific genes.
Proc. Natl. Acad. Sci. USA 82:1074–1078.
26. Tanaka, T., and R. Y. Yada. 1996. Expression of soluble cloned porcine
pepsinogen A in Escherichia coli. Biochem. J. 315:443–446.
27. Ustunol, Z., and T. Zeckzer. 1996. Relative proteolytic action of milk-clotting
enzyme preparations on bovine ␣-, ␤- and ␬-casein. J. Food Sci. 61:1136–
1159.
28. van den Berg, J. A., K. J. van der Laken, A. J. J. van Ooyen, T. C. H. M.
Renniers, K. Rietveld, A. Shaap, A. J. Brake, R. J. Bishop, K. Schultz, D.
Moyer, M. Richmand, and J. R. Shuster. 1990. Kluyveromyces as a host for
heterologous gene expression: expression and secretion of prochymosin.
Bio/Technology 8:135–139.
29. Wang, W., and B. A. Malcolm. 1999. Two-stage PCR protocol allowing
introduction of multiple mutations, deletions and insertions using
QuikChange TM site directed mutagenesis. Bio/Techniques 23:680–682.
30. Zapparoli, G. A., M. Zanazzi, C. Bertezzolo, U. Fantuzzi, P. G. Bianchi, M.
Forini, D. Melani, A. Negrini, and A. De Battisti. 1992. Prove di caseifica-
zione a formaggio grana mediante coagulante chymogen. Latte 17:214–221.