CLONA Y EXPRESA PEPSINÓGENO

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2588–2595 Vol. 70, No. 50099-2240/04/$08.000 DOI: 10.1128/AEM.70.5.2588–2595.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and ItsExpression in Microbial Cells

Rosario Munoz,1 Jose L. Garcıa,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 Biologicas (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 mainprotease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of thisenzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactiveprecursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding forbovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence taglibraries 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 producedfunctional 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 enzymegenerates digestion profiles with -, -, or -casein indistinguishable from those obtained with a naturalpepsin 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 thatthere 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 anacidic 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 siteand maintains the enzyme in its inactive form. The conversionof pepsinogen to active pepsin is initiated by acidic conditionsand involves several conformational changes and bond cleav-age steps that result in proteolytic removal of the prosegmentand release of the active site (20).

The main industrial application of bovine pepsin is in cheesemaking; this enzyme is either naturally present in calf stomachsor is added as a less expensive complement in calf chymosinpreparations (2, 6). The fluctuations in the availability andprice of calf rennet some years ago stimulated the search foralternative milk coagulants; this search included cloning andexpression in several microorganisms of a cDNA coding for

calf prochymosin (3, 17, 28). Recombinant bovine chymosinwas first commercialized in 1990 (5); sales of this compoundquickly accounted for more than one-half of the market forrennet, and several versions, produced from genetically engi-neered Escherichia coli, Kluyveromyces lactis, or Aspergillus ni-

ger , became available. The recombinant rennets have beenshown to be equivalent to natural rennet (18). After the bovinespongiform encephalopathy crisis in Europe, food safety con-

siderations also supported the use of recombinant rennet in-stead of natural rennet. Whereas milk safety has been clearlydocumented 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), thesafety of the abomasum from ruminants is inferred based partiallyon 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, theoff-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 acidificationand in part from the residual proteolytic activity of theenzymes used for milk clotting. The main changes attribut-

able to proteolysis during cheese ripening are changes in thetexture and flavor. The effect on flavor is due to the releaseof peptides and amino acids, which, in turn, can be thesubstrates 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 strategieshave been proposed for accelerating this process in order toreduce 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-

* Corresponding author. Mailing address: Department of Microbi-ology, Instituto de Fermentaciones Industriales (CSIC), Juan de laCierva 3, 28006 Madrid, Spain. Phone: 34-915622900, ext. 359. Fax:34-915644853. E-mail: [email protected].

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ably to the quality of cheeses made with natural rennetpreparations containing pepsin (30).

Several pepsin-encoding genes, including the genes frompigs (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 beencompletely described so far. Only the N-terminal amino acidsequence of bovine pepsinogen (10) and a partial genomicsequence 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 bovinepepsinogen and expression of this cDNA in bacterial and yeastcells. 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 DH5F

[F endA1 hsdR17 (rkmk

) 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 lacI q lacZM15)

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 ml1. 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

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 performedby electroporation (22), and the transformants were selected on LB medium

supplemented with ampicillin (100 g ml1). 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 ml1) 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

TABLE 1. Sequences of the primers used in this study for PCR amplification or for site-directed mutagenesis

Primer Sequence

cDNA-PB3...................................5-CCCGGGCTGCAGAATTCATGTCTGTTGTCAAGATCCCACTCGTCAAAAAGAAGTCC-3cDNA-PB2...................................5-GTGGCGGCCTCCCGGATG-3Xba-PB.........................................5-GCTCTAGAGGGTATTAATAATGAGCGTCGTCAAAATCCCACTCG-3Hind-PB .......................................5-GCGAAGCTTAGTTAGCTATTAGGCCACGGGAGCCAGGCCG-3BP05A ..........................................5-GGAATATTAAGCTTGGTACCGAGCTCGAATTCATGAGATTTCCTTCAATTTTTACTGC-3

BP05B...........................................5 -GATCTTGACAACAGACATAGCTTCAGCCTCTCTTTTATCC-3PT7-PB.........................................5-GGTTTCCCTCTAGAAATAATTTGTTTAACTTTAAGAAGGAGATATACATATGAGCGTCGTCAA-3

VOL. 70, 2004 RECOMBINANT BOVINE PEPSINOGEN PRODUCTION 2589



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 ml1. 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 gelelectrophoresis (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 potentiallycoding 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 containedthe 3 end of the cDNA, including the poly(A) tail. Compari-son of the hypothetical sequence of the cDNA clones with thesequence described previously for the first 110 amino acids of pepsinogen (10) revealed that the clones from theMARC3BOV 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 usedto 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. ADNA fragment containing the sequence coding for bovinepepsinogen that is missing in clone 1Abo04B07 was generatedby PCR performed with primers cDNA-PB2 and cDNA-pB3by using MARC3BOV 103I10 as the template. Primer cDNA-PB3 contains the ATG start codon and the sequence coding forthe four N-terminal amino acids of pepsinogen. This PCRfragment was then inserted into 1Abo04B07 by using aQuikChange kit from Stratagene, as described in Materials andMethods. The resulting plasmid, pBP01, contained a cDNAsequence coding for a complete version of bovine pepsinogenin the phagemid vector pBluescript SK().

Sequence analysis. The peptide sequence of bovine pepsino-gen is 55% identical to the calf prochymosin sequence and 83

and 82% identical to the pig and human pepsinogen se-quences, respectively. The levels of similarity are 94% for pigpepsinogen, 91% for human pepsinogen, and 71% for calf prochymosin. These values are very similar to those obtainedwith the corresponding mature peptides. Two copies of theconsensus sequence for eukaryotic aspartic proteases, flankingthe aspartate residues of the catalytic site as described inPROSITE entry PS00141 (24), are present in the deducedsequence of bovine pepsinogen at positions 75 to 86 and 258 to269. Remarkably, although the N-terminal sequence of thecDNA is identical to the known sequences of pepsinogengenes, there are several differences between the previously

FIG. 1. Sequence alignment showing the experimentally deter-mined N-terminal sequence of bovine pepsinogen (BovPgn) (accessionnumber P00792), the hypothetical translated sequences of cDNA

clones 1Abo04B07 (04B07) (accession number BG937636) andMARC3BOV 103I10 (103I10) (accession number BM106242), thesequence of rabbit pepsinogen III (RabPgn) (accession numberAAA85370), 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 andrabbit pepsinogen III sequences and the bovine pepsinogen cDNAclone sequence are underlined in the bovine pepsinogen exon 6, 7, and8 sequence. The conserved positions for the introns in the genomicsequence for most mammalian aspartic protease genes are indicated byvertical 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 determinedin this work because the sequence in the GenBank database is a partialsequence.

2590 MUNOZ ET AL. APPL. ENVIRON. MICROBIOL.





and primer PT7-PB. This primer provides the original ribo-some binding site from pT7-7. The amplification product wasdigested with DpnI to degrade the DNA from the originalplasmid, pBP02, and the amplified product was transformedinto the expression strain E. coli JM109(DE3). The resultingplasmid, pBP06, contained bovine pepsinogen cDNA undercontrol of the T7 RNA polymerase-inducible 10 promoter.

Several conditions were tested to induce bovine pepsinogensynthesis by E. coli JM109(DE3) cells harboring recombinantplasmid pBP06. The values obtained for pepsinogen activitywere 2 orders of magnitude greater than the values obtainedfor pBP03-transformed cells under similar culture conditions(Table 2). Overnight (16-h) induction at 20°C resulted in themaximum 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 conspicuous40-kDa protein band. We found that most, if not all, of theprotein was recovered from the supernatant of the cell extracts

(Fig. 3B, lane 2), indicating that it was in a soluble form. The40-kDa protein was absent in the supernatants prepared fromeither an uninduced bacterial culture (Fig. 3B, lane 1) or E. coli

cells harboring only the vector plasmid (data not shown). Weobserved a faint band of the same size with cell extracts fromcultures induced for 4 h at 20 and 37°C (data not shown). In

the case of the culture induced overnight at 37°C, we observedan apparent 40-kDa band in the total cell extract, indicatingthat the protein was in an insoluble form, probably inclusionbodies, 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 functionalpepsinogen were recovered in cultures induced at 20°C,whereas no functional protein was detected in cultures inducedat 37°C. Several unsuccessful attempts were made to recoverthe functional protein from the inclusion bodies, based on theprocedure described for recombinant bovine chymosin (4).

Expression of bovine pepsinogen in S. cerevisiae. To producerecombinant bovine pepsinogen in S. cerevisiae, a SacI/XhoIfragment from pBP01 containing pepsinogen cDNA wascloned into pYES2, a yeast episomic expression vector, whichwas digested with the same enzymes. The resulting plasmid,pBP04, contained the bovine pepsinogen cDNA under controlof 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 theculture broth or cell extracts from pBP04-containing yeast cellsthat 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-factorsecretion signal was PCR amplified from S. cerevisiae BY4741genomic DNA by using primers BP05A and BP05B. The re-sulting PCR product was used to introduce the secretion signalinto pBP04, which was fused upstream of the sequence codingfor bovine pepsinogen, with a QuikChange kit from Strat-agene, as described in Materials and Methods. The resultingplasmid, pBP05, and pBP04 were introduced into S. cerevisiae

BY4741 by transformation. The transformants were tested forpepsinogen production and secretion under different inductionconditions (Table 3). Only yeast strains harboring plasmidpBP05 secreted pepsinogen into the culture medium. As ex-pected, the amount of pepsinogen secreted was larger under

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. Thepositions of molecular mass markers (SDS-PAGE standards; Bio-Rad)are indicated on the left.

TABLE 2. Proteolytic activities in cell extracts of E. coliJM109(DE3) transformed with pBP06 under different IPTG

induction conditions

Conditions

Proteolytic activitiesa

20°C 37°C

4 h 16 h 4 h 16 h

Noninduced 0.7 0.2 0.5 0.2 0.3 0.3Induced 4.5 1.3 43.0 7.0 0.3 0.3

a The activities are expressed in milligrams of porcine pepsin equivalents pergram (dry weight). The data are means standard deviations for three separateexperiments.

TABLE 3. Proteolytic activities in the supernatants of S. cerevisiaestrains transformed with different plasmids

MediumProteolytic activitiesa

pYES2 pBP04 pBP05

SD 0.2 0.2 0.2SG 0.2 0.2 0.9 0.3

YPD 0.2 0.2 0.3 0.1YPG 0.2 0.2 1.9 0.5

a The activities after 72 h of induction in different media (SD, SG, YPD, andYPG media) are expressed in milligrams of porcine pepsin equivalents per gram(dry weight). The data are means standard deviations for three separateexperiments.




galactose induction conditions (SG or YPG medium) thanunder glucose repression conditions (SD or YPD medium).However, the yield was clearly increased by using complexmedium (YPG medium). A very low level of pepsinogen pro-duction was detected in cell extracts, and only induced culturescontaining cells carrying plasmid pBP05 had intracellular pep-sinogen levels slightly greater than the background level (datanot shown). There were no significant differences in intracel-

lular pepsinogen levels between cells carrying pYES2 and cellscarrying pBP04. The time course of induction on YPG mediumfor 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 bovinepepsinogen 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 addedto consecutive wells starting with an azocasein hydrolytic ac-tivity equivalent to 4 g of commercial porcine pepsin. Figure5 shows the results of this experiment, which indicated that theclotting 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 andcompared the activities under similar conditions. We studiedthe extent and profile of hydrolysis of purified -, -, and-caseins (Sigma-Aldrich) by using Stabo 230 or acid-activatedrecombinant pepsinogen solutions. The enzyme solutions werepreviously adjusted to contain similar proteolytic activitiesagainst 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 secretedby S. cerevisiae. Both proteases showed the same peptide pat-

tern for the three substrates used, and although the extent of

hydrolysis with the recombinant protein was less than that withStabo 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 thecomplete cDNA sequence encoding true bovine pepsinogenwas cloned and determined for the first time. This conclusionwas based on (i) the identity between the deduced proteinsequence 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 digestionprofiles with the profiles produced by natural bovine pepsin. Inspite of the puzzling noticeable differences between the par-tially described sequences of bovine pepsinogen exons 6, 7, and8 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 theywere most likely obtained from a contaminated cDNA library.It should be stressed that Lu et al. (14) failed to clone thebovine chymosin gene from their library, even when they usedthe homologous probe. In our case, we eliminated contamina-tion since the cDNA fragments described here were derivedfrom two different bovine cDNA libraries and are identical inthe shared regions. Moreover, the hypothetical sequence trans-lated from the cDNA sequence is identical to the first 110amino 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 expressionresults were obtained by using strain JM109(DE3) as the host,pT7-7 as the expression vector, and overnight induction withIPTG at 20°C. Under these optimal conditions, pepsinogenproduction in cell extracts, expressed in porcine pepsin equiv-alents, was more than 40 mg/g (dry weight). The highest levelsof 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 mediumwith galactose as the only carbon source (YPG medium) forinduction. Even though the yeast expression levels were lower

than those inE. coli

, the use of pBP05 has the advantage thatthe recombinant pepsinogen is recovered from the culturebroth. The secretion of the molecule facilitates both purifica-tion and production in a continuous culture system.

The lack of pepsinogen production in yeast carrying plasmidpBP04 may have been due to transcriptional problems. In thisplasmid the distance between the transcription start site andthe start codon is greater than the distance in pBP05; this isdue to the presence of pBluescript multiple-cloning-site se-quences that have not been removed. Nevertheless, we cannotexclude 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-

FIG. 5. Milk-clotting activity of native pepsinogen (Stabo 230) orrecombinant pepsinogen obtained from yeast strains carrying plasmidpBP05. The amount of enzyme used in each column is expressed inmicrogram equivalents of porcine pepsin.

FIG. 4. Time course of extracellular pepsinogen formation in yeastcultures transferred to YPG induction medium. Symbols: F, pYES2-transformed cells; s, pBP05-transformed cells.




nant bovine chymosin (4) have been unsuccessful. However, if the large amounts of protein obtained as inclusion bodies incultures 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 improvingyield and purity.

One of the main potential applications of the recombinantenzyme is to accelerate ripening in cured cheese or to producecheeses with properties similar to those made with rennet,which naturally contains both chymosin and pepsin. Indeed, ithas been shown that ripening of Grana cheese is accelerated orimproved by using mixtures containing 10% bovine pepsincompared 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 preparationswere 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 milkclotting 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 werealso similar in terms of the degree of proteolysis and the mainhydrolysis products. Only in the case of -casein was there asmall difference in the degree of hydrolysis, and this was prob-ably due to slightly different amounts of enzyme used for theassay. 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 theincubation temperature, and the protein is degraded morerapidly at 2°C than at 32°C. It has been suggested that pepsinis capable of degrading both - and -caseins but is unable todegrade -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-

binant bovine pepsinogen, which generated the same peptideprofile.

In conclusion, this is the first time that a recombinant bovinepepsinogen has been synthesized, and our findings pave theway for using this enzyme as an alternative in cheese making;use of this recombinant enzyme has the advantage that therecombinant pepsin should be free from potential pathogenicagents arising from animal tissues, particularly the causativeagent of bovine spongiform encephalopathy. In addition, likerecombinant bovine chymosin, recombinant pepsin would bemore acceptable than the native enzyme to vegetarian consum-ers or to people subject to food restrictions due to religiousbeliefs. Finally, other potential applications of bovine pepsininclude 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 providingcDNA clones from the 1Abo library, to E. Cebollero for help withyeast transformation, to F. Jorganes and A. Fernandez for technicalassistance, and to M. Sheehan for correcting the English version.

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CLONA Y EXPRESA PEPSINÓGENO