Gene, 21{ 1984) 35-46 Elsevier 35 GENE 934 Expression of calf prochymosin in S~cc~u~~~yce~ cwev&iae (Recombinant DNA; shuttle vector; rennin; baker’s yeast; GAL1 promoter; milk-clotting) Christopher G. GofP, Donald T. Moir **, Tadahiko Kohno, Thomas C. Gravius, Robert A. Smith, Edith Yamasaki and Alison Taanton-Rigby Department of Molecular Genetics, Collaborative Research, Inc.. Lexington, MA 02173 (U.S.A.) Tel. (617) 861-9700 (Received July 21st, 1983) (Revision received October 17th, 1983) (Accepted October 20th, 1983) SUMMARY A yeast strain which synthesizes activatable calf prochymosin (also known as prorennin) has been constructed by transformation with a vector carrying the methionyl-prochymosin coding sequence attached to efficient yeast ~~sc~ption~ promoter and te~nator sequences. Cloned pr~roch~os~ cDNA was altered by restriction endonuclease cleavage and addition of a synthetic oligonucleotide to yield a DNA sequence encoding methionyl-prochymosin. This methionyl-prochymosin gene was ligated to a yeast chromosomal fragment containing the GAL1 promoter, and the construction was placed in an Escherichia coli-Saccharomyces cerevisiae shuttle vector with or without a transcriptional terminator DNA fragment from the yeast WC2 gene. In yeast the two constructions result in equal amounts of prochymosin protein and mRNA. The proch~osin from yeast is activatable to chymosin by incubation at low pH and exhibits milk-clotting activity indistinguishable from calf chymosin. INTRODUCTION The ability to transform the yeast S. cerevisiae with autonomously replicating plasmids and the availabil- ity of many cloned yeast genes and promoter seg- ments have made that organism an attractive host for * Present address: Biology Department, Haverford College, Haverford, PA 19041 (U.S.A.) Tel. 215-896-1000. ** To whom correspondence should be addressed. Abbreviations: bp, base pairs; A, deletion; kb, kilobases or kilobase pairs; p, plasmid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; T,,,, melting temperature ofdouble-stranded nucleic acid. expression of heterologous genes. For example, several cl-interferon genes (Hitzeman et al., 1981; Tuite et al., 1982), the pinterferon gene (Derynck et al., 1983), and the hepatitis B surface antigen (Valenzuela et al., 1982; Miy~oh~a et al., 1983) have recently been expressed in yeast. Unlike E. co&, yeast produces no endotoxins; therefore, adequate purification of pharmaceutical products should be more easily accomplished. Yeast should also be an ideal host organism for production of proteins involved in food processing because it is a common constituent of many diets. For example, we are particularly interested in the microbiological production of calf chymosin, a milk- 0378-l 119/84~SO3.00 0 1984 Elsevier Science Publishers
Transcript
Gene, 21{ 1984) 35-46 Elsevier
35
GENE 934
Expression of calf prochymosin in S~cc~u~~~yce~ cwev&iae
Christopher G. GofP, Donald T. Moir **, Tadahiko Kohno, Thomas C. Gravius, Robert A. Smith, Edith Yamasaki and Alison Taanton-Rigby
Department of Molecular Genetics, Collaborative Research, Inc.. Lexington, MA 02173 (U.S.A.) Tel. (617) 861-9700
(Received July 21st, 1983) (Revision received October 17th, 1983) (Accepted October 20th, 1983)
SUMMARY
A yeast strain which synthesizes activatable calf prochymosin (also known as prorennin) has been constructed by transformation with a vector carrying the methionyl-prochymosin coding sequence attached to efficient yeast ~~sc~ption~ promoter and te~nator sequences. Cloned pr~roch~os~ cDNA was altered by restriction endonuclease cleavage and addition of a synthetic oligonucleotide to yield a DNA sequence encoding methionyl-prochymosin. This methionyl-prochymosin gene was ligated to a yeast chromosomal fragment containing the GAL1 promoter, and the construction was placed in an Escherichia coli-Saccharomyces
cerevisiae shuttle vector with or without a transcriptional terminator DNA fragment from the yeast WC2 gene. In yeast the two constructions result in equal amounts of prochymosin protein and mRNA. The proch~osin from yeast is activatable to chymosin by incubation at low pH and exhibits milk-clotting activity indistinguishable from calf chymosin.
INTRODUCTION
The ability to transform the yeast S. cerevisiae with autonomously replicating plasmids and the availabil- ity of many cloned yeast genes and promoter seg- ments have made that organism an attractive host for
* Present address: Biology Department, Haverford College, Haverford, PA 19041 (U.S.A.) Tel. 215-896-1000. ** To whom correspondence should be addressed.
Abbreviations: bp, base pairs; A, deletion; kb, kilobases or kilobase pairs; p, plasmid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; T,,,, melting temperature ofdouble-stranded nucleic acid.
expression of heterologous genes. For example, several cl-interferon genes (Hitzeman et al., 1981; Tuite et al., 1982), the pinterferon gene (Derynck et al., 1983), and the hepatitis B surface antigen (Valenzuela et al., 1982; Miy~oh~a et al., 1983) have recently been expressed in yeast. Unlike E. co&,
yeast produces no endotoxins; therefore, adequate purification of pharmaceutical products should be more easily accomplished.
Yeast should also be an ideal host organism for production of proteins involved in food processing because it is a common constituent of many diets. For example, we are particularly interested in the microbiological production of calf chymosin, a milk-
making. Two groups have recently reported isolation
of cDNA clones containing the coding information
for preprochymosin (Moir et al., 1982; Harris et al.,
1982) a precursor form of chymosin containing a
16-amino acid signal peptide at the amino-terminal
end of the zymogen prochymosin. Isolation of
chymosin from its natural source, the fourth stomach
of an unweaned calf, actually yields a mixture of
prochymosin and chymosin, each existing as two
types called A and B (Foltmann, 1962). The amino
acid sequence of the two types was shown to differ
in at least one residue (Foltmann et al., 1979). Work
from this laboratory, demonstrating that there is a
single preprochymosin gene per haploid genome,
indicates that the A and B types must represent
products of different alleles in the population (Moir,
et al., 1982). While the reported differences in the
properties of the A and B types of chymosin are
minor (Foltmann, 1970), prochymosin and chymo-
sin themselves differ in a number of properties. For
example, studies on purified preparations of calf
chymosin and prochymosin have shown that above
pH 5, prochymosin is more stable than chymosin,
and that below pH 5, prochymosin is converted to
the active form, chymosin, by an apparent auto-
catalytic proteolysis which removes an amino-ter-
minal 42-residue peptide from the zymogen. Nothing
is known about the properties of preprochymosin
since it has never been isolated from the calf. There-
fore, because of its stability and because it can be
readily converted to the active species, prochymosin
is probably the most suitable form of the enzyme to
produce in yeast.
We report here the construction of a DNA se-
quence coding for methionyl-prochymosin A and
expression of that gene in yeast by using the highly
regulated yeast GAL1 gene promoter. Regulation of
prochymosin expression from this promoter is
shown to be controlled by the carbon source at the
level of transcription and/or mRNA stability, as is
known for expression of the yeast GALI gene which
encodes galactokinase (St. John and Davis, 1981).
Promoter function as measured by the steady-state
level of prochymosin mRNA is quite efficient. Trans-
lation of that mRNA in yeast yields a protein indis-
tinguishable from authentic calf prochymosin by
criteria of size, immunoreactivity and biological
activity.
MATERIALS AND METHODS
(a) Strains and media
E. coli strains, growth media and recombinant fl
bacteriophage R207 and R118/37 carrying the com-
plete preprochymosin coding sequence were pre-
viously described (Moir et al., 1982). The fl phage
CGF12 containing unique XbaI and EcoRI sites was
created from fl phage R229 (Zinder and Boeke,
1982) by restriction with EcoRI, incubation with
E. coli DNA polymerase I Klenow fragment plus all
four deoxynucleoside triphosphates to fill in cohesive
termini, and ligation with an X&I synthetic oligonu-
cleotide linker (dCCATCTAGATGG). An isolate
which had fortuitously lost one of the expected flank-
ing EcoRI restriction sites was identified and kept as
CGF12 (G. Vovis, unpublished results).
The galactose-utilizing yeast host strain was
CGY150 (MAT a ura3-52 leu2-3). Yeast transfor-
mation was by the spheroplasting method of Hinnen
et al. (1978) or the LiCl method of Ito et al. (1983).
Plasmid-containing yeast strains were grown in min-
imal medium (0.67% yeast nitrogen base, Difco)
with leucine (50 pg/ml) and carbohydrate, either glu-
cose or galactose, at 2%. Strain CGY 189 is
CGY 150 containing plasmid pCGS 128, CGY238 is
CGY150 with pCGS168, and CGY461 is CGY150
with pCGS242.
Plasmid YCpSO-Sc4816 d AUG (Fig. 2) was a gift
from R. Davis (Stanford Univ.) and contains an
engineered yeast chromosomal fragment carrying the
promoters for yeast genes GAL1 and GAL1 0 inserted
into the yeast-E. coli shuttle vector YCp50. The
plasmid was constructed by BAL31 exonuclease
trimming from a natural restriction site in the GAL1
structural gene followed by ligation with BamHI lin-
kers; the resulting unique BamHI site is four nucleo-
tides upstream from the normal position of the GAL1
initiation codon (Thomas, M., Johnston, M. and
Davis, R.W., manuscript in preparation).
(b) Enzymes and synthetic oligonucleotides
Restriction enzymes were from New England
Biolabs, T4 DNA ligase and T4 polynucleotide
kinase from Collaborative Research, Inc., and E. coli
DNA polymerase I Klenow fragment and S 1 nucle-
ase from Boehringer. Enzymes were used as recom-
37
mended by the supplier. Hemoglobin mRNA (80% pure) was from BRL.
Synthetic oligonucleotide linkers X&I (dCC
ATCTAGATGG), Sal1 (dGGTCGACC) and Hind111 (dCCAAGC’I”TGG) were synthesized at Collaborative Research, Inc.
(c) Isolation of yeast protein and RNA
Yeast strains were grown on the appropriate mini- mal medium with selection for the presence of plas- mid (where necessary) by demanding uracil proto- trophy. At a density of Klett 50 (about 2 x 10’ cells/ml) cells were harvested by centrifugation, washed with water and stored at -70’ C.
Total cellular RNA was isolated essentially by the method of Russell and Hall (1982). RNA gel-transfer hybridization was performed as follows. Total yeast RNA (5 pg per gel lane) was treated with glyoxal according to the method of McMaster and Carmi- chael (1977), subjected to electrophoresis in a 1 y0 agarose gel, transferred to nitrocellulose, probed with 32P-labeled nick-translated double-stranded fl phage R118/37 DNA (2.5 x lo7 cpm/pg, 1.7 x lo6 cpm/lane) and washed exactly as described by Thomas (1980).
Yeast proteins for gel-transfer immunoassay were isolated by either of two methods. According to method A, cell pellets (about lo9 cells) were vortexed with 0.3 g glass beads as described by Rose et al. (1981), except that the buffer (250 ~1) was 62.5 mM Tris * HCl (pH 6.8) and 10% glycerol. After centrifu- gation at 12000 x g for 5 min to remove cellular debris, the prochymosin in the supernatant behaved as a soluble monomer of the expected size (M, approx. 40000) upon gel filtration on Sephadex G-100. For gel-electrophoretic analysis, the super- natant was treated with SDS to a final concentration of 1 y0 and with 2-mercaptoethanol to 0.7 M. The mixture was rapidly heated to 100°C for 3 min and a portion (20-300 fig protein) was applied to an SDS polyacrylamide gel (Laemmli and Favre, 1973).
According to method B, the same amount of yeast cell pellet was resuspended in 1 ml H,O and 0.2 ml of 100 y0 (w/v) TCA solution was added. The mixture was vortexed for 3 to 4 min with 1.5 g glass beads as described above, then diluted by addition of 1 ml of 5 % (w/v) TCA solution. This mixture was centrifug- ed 10 min at 13000 rev./min. The supernatant was
discarded and the pellet, including the glass beads, was resuspended in 0.1 ml of buffer containing 5 y0
SDS, 80 mM Tris * HCl (pH 6.8), 12.5% glycerol, 4 % 2-mercaptoethanol and 0.00 1% bromphenol blue. After neutralization with 1 M Tris base, the mixture was heated in a boiling water bath for 10 min and centrifuged 15 min at 18000 rev./min. The su- pernatant, which contains 100% of the prochymosin in yeast cells carrying the appropriate expression plasmid, was applied directly to an SDS poly- acrylamide gel as described above.
After electrophoresis, proteins were transferred to nitrocellulose, then probed with anti-chymosin anti- serum (gift of R. Goltz, Dow Chemical Co.) followed by ‘251-labeled Staphylococcus aureus protein A (New England Nuclear, 0.5 @/lane), essentially as described by Towbin et al. (1979). To quantitate the amounts of prochymosin obtained from yeast, auto- radiographs having signals from yeast prochymosin and known amounts of calf prochymosin were scan- ned on a Beckman DU-6 spectrophotometer equip- ped with a film-scanning accessory. Calf chymosin and prochymosin standards were purified from the calf abomasum essentially as described by Foltmann (1970) and supplied by R. Goltz of Dow Chemical co.
(d) Nuclease mapping of RNA transcripts
Transcription initiation points were determined by a modification of the method of Berk and Sharp (1977). DNA fragments were 5’-end labeled on their antisense strands using [ Y-~~P]ATP (Amersham) and T4 polynucleotide kinase as described by Maxam and Gilbert (1980), then isolated from agarose gels by using a modification of the potassium iodide gel dissolution method (Smith, 1980). Less than 0.1 pg of each labeled fragment was copre- cipitated with 25 pg of total yeast RNA from strain CGY 189 (by addition of ethanol to 70%) and resus- pended in 100 ~1 of 70 % formamide, 100 mM PIPES (pH 6.8), 10 mM EDTA, 0.35 M NaCl. This mix was denatured at 80°C for 10 min and allowed to reanneal for 1 h at each of the following tempera- tures, in sequence: 63°C 55°C 49°C and 44°C since the exact T, of DNA-RNA hybrids in this buffer was not known. After two cycles of ethanol precipitation, samples were resuspended in 100 ~1 5% glycerol, 30 mM sodium acetate (pH 4.6),
50 mM NaCl, 1 mM ZnSO, and treated with 25 units of mung bean nuclease (P-L Biochemicals) for 1 h at 37°C. Following phenol extraction and etha- nol precipitation, the sizes of protected DNA frag- ments were determined by comparison to fragments of known size on 7 y0 polyacrylamide 7 M urea gels.
(e) Milk-clotting assays
Samples were tested for milk-clotting activity in an assay modified from Foltmann (1970). The total volume of milk was 1 ml and enzyme samples of 100-200 ~1 in 0.05-O. 1 M sodium phosphate (pH 6) were added. Prochymosin was activated by titration to pH 2 with lactic acid and incubation at room temperature for 30 min before neutralization with NaOH and assaying (Rand and Ernstrom, 1964).
(f) Partial puri~cation of prochymosin from yeast
Prochymosin was partially purified from 800 ml of CGY461 culture grown to mid-exponential phase (about 2 x lo7 cells/ml) in minimal medium supple- mented with leueine (50 pg/ml) and galactose (2%). Cells were broken at O-4’ C in 4 ml of 0.1 M sodium phosphate buffer (pH 5.8) by vortexing with 2/3 vol. of acid-washed glass beads (0.3-0.5 mm). The crude lysate was removed from the settled glass beads and centrifuged at 13000 rev./min for 30 min. The clari- tied supe~atant was loaded onto a DE52 (What- man) column (1 x 27 cm) equilibrated with 0.1 M sodium phosphate (pH 5.8), and prochymosin was eluted with a linear gradient of 150 ml 0.1 M sodium phosphate (pH 5.8) and 150 ml 0.3 M sodium phos- phate (pH 4.3) essentially as described by Foltmann (1962). Fractions ~ont~ning proch~osin were identified by the immunological method of Towbin et al. (1979) described above and pooled. We ob- served a seven-fold purification of the soluble pro- chymosin extracted by this method and a recovery of about 30% of the milk-clotting activity.
RESULTS
(a) construction of the ATG-prochymosin coding sequence
In order to construct a calf prochymosin coding sequence which can be expressed in heterologous
cells, the nucleotide sequence encoding the sixteen amino acid signal sequence of preprochymosin was removed and replaced with an ATG initiation codon.
DoubIe-stranded replicative form DNA from re- combinant fl phage R207 was cut with Hip2- dII1 + SgZII to generate a fragment (435 bp) containing the beginning of the preprochymosin, prochymosin and chymosin coding regions (Fig. 1). Access to the beginning of the prochymosin coding region was gained by incomplete cleavage of the above fragment with HhaI to generate a 180-bp H/z&to-BglII fragment. The 3’ HhaI overhang in- cluding the initial G residue of the first alanine codon of pro~hymosin was removed by incubation with DNA polymerase I Klenow fragment. A self-com- plementary synthetic oligonucleotide of sequence S’dCCATCTAGATGG 3’ was ligated to the flush ends of the fragment; this oligonucleotide provides the missing G residue for the first codon of authentic prochymosin, an ATG initiation codon, and an XbaI restriction endonuclease site. The fragment was di- gested with X&I and cloned in fl phage vector CGF 12 which contains unique XbaI and EcoRI sites (see MATERIALS AND METHODS, section a) to yield phage CGF2 1 (Fig. 1). The remainder of the prochy- mosin gene was obtained from recombinant fl phage R118/37. The entire gene was regenerated by using the unique &I site in the prochymosin-coding DNA and was cloned in pBR322 to yield pCGE68 (Fig. 1).
(b) Construction of a plasmid designed for expression
of prochymosin in yeast
Virtually aI1 yeast genes which have been sequenc- ed contain an A residue at position - 3 (A of the ATG defines position + 1) and a T at position + 6 (Dobson et al., 1982). The ATG-prochymosin cod- ing sequence in pCGE68 already contains a T residue at + 6 but also has a T at position - 3. Accordingly, the plasmid was cut with XbaI, the 5’ overhangs were trimmed away with Sl nuclease to gain access to the ATG codon, and a SaZI linker (5’ dGGTCGACC 3’) was ligated on to provide an A residue at position - 3 (see Fig. 2). The recircular- ized plasmid, containing the S&I linker, is designated pCGE91.
The promoter for the construction was derived from a fragment of yeast chromosome II which contains the divergent promoters for the GAL1 and
39
1 Hind Ill
Hind Hha P Hha ee’
4 4 4
pre “P chymosin
Pst I
Hind Ill
GAL10 genes cloned as plasmid YCpSO- Sc48 16dAUG (MATERIALS AND METHODS, section a). The plasmid was cleaved by BarnHI, which cuts at an engineered site four nucleotides 5’ to the normal position of the initiator ATG codon of the yeast GAL1 (galactokinase) gene (Fig. 2). The BumHI ends were blunted by treatment with DNA polymer- ase I Klenow fragment in the presence of all four deoxynucleoside triphosphates; finally the 850-bp fragment containing the GAL1 promoter as well as the GAL10 promoter plus about 150 bases of trans- cribed GAL10 sequence was released by digestion with EcoRI.
The vector for the construction was derived from the plasmid YIp5 (Botstein et al., 1979) by addition of a yeast origin of replication. The 2.2-kb EcoRI fragment of form B 2~ DNA isolated from plasmid YEp21 (Botstein et al., 1979) was further cut with HpaI and HindIII, treated with DNA polymerase I Klenow fragment in the presence of all four
Fig. 1. Construction of an ATG-prochymosin gene. Single lines
deoxynucleoside triphosphates, and inserted into the PvuII site of YIp5 (Fig. 2). The resulting vector pCGS40 is a “shuttle vector” because it contains origins of replication which function in E. coli (the pBR322 on’ region from YIPS) and S. cerevisiue (the yeast 2~ fragment), plus markers selectable in E. coli
(the pBR322 amp’ gene from YIp5) and S. cerevisiue
(the yeast URA3 gene from YIPS). The 2~ DNA in pCGS40 lacks the EcoRI, HindIII, PvuI and PstI sites present in the larger 2~ fragment in other shuttle vectors such as YEp21; therefore, it is more con- venient for subcloning manipulations.
The promoter, prochymosin gene, and vector DNAs were assembled in a three-molecule ligation. This reaction contained the EcoRI to BamHI (blunt- ed) GAL1 promoter fragment as described above, a SalI (blunted with DNA polymerase Klenow frag- ment and all four deoxynucleoside triphosphates) to Hind111 fragment from pCGE91 containing the ATG-prochymosin coding sequence, and the larger
40
a
b
Fig. 2. Construction of a yeast expression vector for methionyl- prochymosin. (a) Diagram of the assembly strategy. Single lines denote pBR322 sequences and double lines represent yeast DNA and the calf prochymosin cDNA gene (MATERIALS AND METHODS, section a). Restriction endonuclease recognition sites are as in Fig. 1.; Sal1 {S). (b) Comparison of the nucleotide sequences of the GAL1 promoter-g~actokinase gene junction (upper line) and the GAL1 promoter-prochymosin gene junction (lower line). BumHI and Sal1 refer to non-functional residual linker sequences.
fragment of pCGS40 produced by digestion with EcoRI and HndIII. The resulting plasmid pCGS 128 (Fig. 2) should direct the synthesis of calfprochymo- sin in S. cerevisiae.
Plasmid pCGS 128 contains no yeast tr~sc~ption termination signals other than those which might fortuitously be present farther downstream from the prochymosin sequence in the URA3 and 2~ DNA. Therefore, an efficient transcription terminator was added to pCGS128 at a site close to the end of the proch~osin coding region. Plasmid pRB58 contains the yeast SUC2 gene (which codes for invertase) derived from a partial Sau3A library of yeast chromosomal DNA (Carlson and Botstein,
1982). A 900-bp fragment containing the end of the invertase coding DNA and the site of transcription termination was obtained by (1) restriction with HpaI, which cleaves within the SK2 structural gene; (2) addition of aHind synthetic oligonucleo- tide linker (5’ dCCAAGCTTGG 3’); and (3) re- striction with HindIII, which cleaves both the linker and a natural Hind111 site downstream from the SUC2 gene. The fragment was inserted into the Hind111 site of plasmid pCGS128 (Fig. 2), and a plasmid carrying the terminator in the normal orien- tation (relative to transcription) was isolated (pCGS 168).
41
(c) Transcription of the ATG-prochymosin gene in yeast
The chymosin-specific mRNA transcripts in yeast from plasmids pCGS 128 and pCGS 168 were exam- ined by gel-transfer hybridization. Total yeast RNA isolated from strains CGY 189 and CGY238, carry- ing plasmids pCGS 128 and pCGS 168, respectively, was denatured with glyoxal, separated by electro- phoresis in an agarose gel, transferred to nitrocellu- lose and probed with nick-translated 32P-labeled phage R118/37 DNA. As shown in Fig. 3, no chy- mosin-specific RNA is observed when cells are grown on glucose {lanes B and D), consistent with the known regulation of the yeast GAL1 gene (St. John and Davis, 198 1). On the other hand, when grown on galactose, strain CGY189 produces two distinct chymosin-specific RNA species about 2.8 kb and 3.8 kb in size (lane A). Galactose-grown cells of strain CGY238 produce a single chymosin- specific mRNA species of about 1.75 kb (lane C). The fact that a single transcript is observed here, together with its reduced size compared to the trans- cripts from pCGS128, indicates efficient trans- cription te~ination in the SUC2 DNA segment of plasmid pCGS 168.
Previous studies on the SUC2 mRNA in yeast have shown that all the transcripts terminate at a unique site about 1.03 kb from the internal BumHI site of the gene (Carlson and Botstein, 1982). In pCGS 168, this site is about 630 bp downstream from the Hind111 site that forms the junction of prochymosin and WC2 DNA. In the present con- structions, pCGS128 and pCGS168, the ATG-pro- chymos~ coding region is 1100 bases long; there- fore, the addition of 630 bases of RNA 3’ to the coding region would produce a transcript of 1.7 kb from pCGS 168, essentially the size observed (Fig, 3, lane C). This result suggests that transcription from the GAL1 promoter begins very close to the start of the ATG-prochymosin gene. To confirm this sug- gestion, nuclease mapping of the prochymosin trans- cription start site in pCGS128 was carried out. An EcoRI-to-BgflI fragment from pCGSl28 spanning the GAL1 promoter and the beginning of the prochy- mosin gene, was isolated after 32P-labeling of the BglII end. This fragment was denatured, hybridized to RNA from strain CGY189, digested with mung bean nuclease, and then examined by polyacrylamide
Fig. 3. Andysis of proch~os~-speci~c transcripts from yeast by RNA gel-transfer hybridization. Total RNA from strain CGY 189 grown on galactose (A), or glucose (B) and total RNA from strain CGY238 grown on galactose (C) or glucose (D) were denatured with glyoxal, electrophoresed, transferred to nitrocel- lulose and probed with [3ZP]DNA. The positions of 2% (3.4 kb) and 18s (1.6 kb) yeast ribosomal RNA on this gel are denoted at left.
gel electrophoresis. The CGY189 RNA protects from nuclease digestion 260 + 10 bases of the labeled DNA fragment (Fig. 4). Since the BgflI site is 184 bp beyond the proch~os~ initia~on codon, the mRNA leader must be 76 + 10 bases long. Trans- cription of the wild-type GAL1 gene appears to initiate at multiple closely spaced points generating
a 65-70-bp leader (Thomas, M., Johnston, M. and Davis, R.W., manuscript in preparation); therefore, the prochymosin transcript from pCGS128 starts very near the normal region of GAL2 start points.
A :’ @
Fig. 4. Mung bean nuclease of the mRNA start point of the
GALI-promoted methionyl-prochymosin gene in yeast strain CGY189 (MATERl,~LS AND METHODS, section d). A
[“P]DNA fragment from plasmid pCGS128 was protected from
mung bean nuclease digestion by total yeast RNA from strain
CGYl89. The protected fragment (identified by arrow at left)
was sized by eIectrophoresis in a polya~ryiamide gel (lane B)
along with labeled DNA fragments of known length (pBR322
digested with A4spI (lane A) or HneII (lane C) and 5’-end labeled
with [Y-~*P]ATP).
The 2.8-kb and 3.8-kb transcripts from pCGSl28 must include about 1.7 kb and 2.7 kb of RNA in addition to the 1100 bases of prochymosin coding information. This would be consistent with the exis- tence of transcription termination sites at each end of the l.l-kb yeast URA3 DNA insert in pCGS 128. Thus, transcription termination, in all cases, appears to occur within yeast DNA sequences on the plas- mid. The steady state level of total chymosin-specific RNA is unchanged regardless of whether trans- cription te~~nation occurs within 630 bases of the end of the prochymosin gene or l-2 kb away (see
Fig. 3). To quantitate chymosin-specific mRNA in
CGY238, known amounts of poly(A)-cont~ning RNA from CGY238 and known amounts of purified globin mRNA were electrophoresed on agarose gels and transferred to nitrocellulose. The nitrocellulose membranes were probed with equivalent amounts of nick-translated 32P-labeled fl phage R118/37 DNA and p/?Gl DNA (which encodes &globin; Maniatis et al., 19764, labeled at equal specific activities. The results indicate that chymosin-specific RNA repre- sents 2 to 5% of the total poly(A)-containing RNA in CGY238 (not shown).
(d) Synthesis of prochymosin in yeast and activation to chymosin
Extracts prepared by method A from yeast strain CGY189 (carrying pCGS128) contain a protein which migrates on SDS polyac~Iamide gels at a position identical to that of authentic prochymosin from the calf and which binds antibodies raised against chymosin (Fig. 5, lane C). Comparison with a known amount of authentic calf prochymosin (lane A) indicates that 0.02% of the soluble protein of strain CGY189 is prochymosin. The same result is obtained for yeast strain CGY238 carrying pCGS 168 (data not shown). Extracts from the same host strain carrying a similar plasmid without the prochymosin gene do not contain an immunological- ly related protein the size of prochymosin (lane B). A smaller immunologically reactive protein is ob- served in all the yeast extracts, but it is apparently a yeast protein which fortuitously cross-reacts with a subset of the antibodies. Pre-absorption of the anti- serum with extracts from CGY 150, the yeast host strain without a plasmid, diminishes the intensity of
43
A D E FG
Fig. 5. Analysis of prochymosin from yeast by gel-transfer immunoassay. All extracts were made by method A (see MATERIALS AND METHODS, section c) unless noted otherwise. Lane A: 300 pg soluble protein from CGYl50 (untransformed host) mixed with 10 ng calf prochymosin (PC) and 30 ng calf chymosin (C) standards; lane B: 300 pg soluble protein from CGY 150 transformed with a plasmid containing no prochymosin sequences; lane C: 300 pg soluble protein from CGY 189; lane D: 10 ng of calf prochymosin standard; lane E: 50 pg protein extracted from CGY461 by method B; lanes F and G: 50 pg and 20 pg of protein extracted from CGY461 by method A; lane H: 10 ~1 (about 5 to 10 ng) of partially purified, fully activated prochymosin from CGY461; lane I: 17 ng calf prochymosin standard; lane J: same as lane H, but mixed with 17 ng calf prochymosin ; lane K: 30 ng calf chymosin standard. The lowest bands in lanes A and K are degradation products of the calf chymosin standard; the intense band across lanes A, B and C is a yeast protein which fortuitously reacts with antibodies in the serum used here (see RESULTS, section d).
that protein band but not the prochymosin-sized band (not shown).
The yeast-synthesized prochymosin was partially purified in order to characterize the protein more thoroughly. For this work, a different strain, CGY461, was derived by transformation of the same host strain CGY 150 with a slightly different plasmid, pCGS242. The plasmid is identical to plasmid pCGS 168 with two exceptions. First, the pCGS 168 2~ origin of replication has been replaced by a 3.3-kb EcoRI fragment containing the yeast LEU2 gene and derived by incomplete EcoRI digestion of plasmid pJDB219 (Beggs, 1978). Second, this fragment has been inserted at the EcoRI site adjacent to the GAL1 DNA (Fig. 2). For reasons still unclear, plasmid pCGS242 directs the synthesis of two- to five-fold
more prochymosin than do plasmids pCGS128 or pCGS168. As shown in Fig. 5, strain CGY461 grown on galactose synthesizes prochymosin at a level of about 0.1% of the soluble protein when extraction is by method A (compare lanes D and G). Extraction by method B results in even more pro- chymosin. Lanes E and F of Fig. 5 each contain about 50 pg of total yeast protein obtained by ex- tracting according to method B or A, respectively. The amount of prochymosin obtained differs by three to five fold. Thus, CGY461 synthesizes approx. 0.5% of its protein as prochymosin; but the use of a chaotropic agent such as trichloroacetic acid is required to efficiently extract most of the prochy- mosin.
In order to examine the biological activity of the
H IJK
44
yeast-synthesized prochymosin, the use of chao- tropic agents and denaturants was avoided and the prochymosin was extracted as described in MATERIALS AND METHODS, section f. The purifi- cation steps included DEAE-cellulose chromatogra- phy and ammonium sulfate precipitation, the same techniques used by Foltmann (1962) for purification of prochymosin from the calf (see MATERIALS AND
METHODS, section f). Yeast-synthesized prochymo- sin was eluted from the DEAE-cellulose column at the same concentration which elutes calf prochymo- sin. Fractions containing prochymosin were identi- fied by reaction with anti-chymosin antibody after SDS gel electrophoresis and transfer to nitrocel- lulose. Peak fractions were pooled, concentrated by ammonium sulfate precipitation, redissolved in 0.1 M sodium phosphate (pH 5.8), and dialyzed against the same buffer.
After incubation at pH 2 for 30 min as is done to activate calf prochymosin (see MATERIALS AND
METHODS, section e), the partially purified prochy- mosin from yeast was completely converted to a protein the size of chymosin (Fig. 5, lane H). Judging from authentic calf chymosin standards on the same gel, the concentration of yeast chymosin in the par- tially purified activated mixture is 0.5-1.0 pgjml (Fig. 5, lanes H and K). The activity of the same partially purified yeast chymosin in a milk-clotting assay (See MATERIALS AND METHODS, SeCtiOn e) iS
104 milliunits/ml. The specific activity of yeast- synthesized chymosin calculated from these data (100-200 units~mg) is within experimental error of the value for pure calf chymosin (100 units/mg; Folt- mann, 1970).
As a control for the purification and activation expe~ments, a parallel fractionation by DEAE-cellu- lose chromatography, followed by pH 2 treatment, was carried out on protein extracted from CGY 150, the host strain containing no plasmid. No milk-clot- ting activity was observed in these control fractions.
DISCUSSION
Efficient expression of a cloned heterologous gene requires, at a minimum, signals for transcription and translation compatible with the host organism. The results presented here show that a yeast chromoso- ma1 fragment containing DNA upstream from the
GALI gene translation initiation codon provides those signals for expression of the calf prochymosin gene in yeast. Transcription of the prochymosin gene in CGY 189 and CGY238 is regulated like the GAti gene, and the prochymosin transcript represents 2-5 y0 of the total poly(A)-containing KNA in induc- ed cells. The prochymosin gene is present in multiple copies in these cells because the vector contains a 2~ origin of replication. Estimates of its copy number
suggest that it is approximately equal to that of YEp21, about 20-30 copies per cell {unpublished observations). Therefore, transcription from a single copy of the plasmid would presumably represent about O.l-0.2% of the cell’s mRNA. Published estimates of the amount of tr~scription produced from a single chromosomal copy of the GAL1 gene are in the range of 0.25-l .O% of the total polyadeny- lated mRNA (St. John and Davis, 1981). From these results it appears that multiple copies of the GAL1 promoter result in increased production of mRNA, but the data do not allow determination of the increments increases in transc~ption level with
increasing copy number. Nevertheless, the absolute steady state amount of prochymosin mRNA in the yeast strains described here should be adequate to support a high level of prochymosin production if translation of that mRNA were as efficient as that of most naturally occurring yeast mRNAs.
In addition, the amount of prochymosin-speci~c RNA is not affected by alterations in the trans- cription termination site. Judging from the compar- able steady state levels of chymosin-specific trans- cripts in CGY 189 and CGY238, mRNA stability is unaffected by increases in length at the 3’ end of one to two kilobases. This result contrasts sharply with previous observations concerning the yeast cycl-5 12 mutation, which is a deletion of the transcription termination signal site resulting in a 1 kb longer cycl
mRNA (&ret and Sherman, 1982). In that case the amount of cycl mRNA dropped to 10% of its normal level. Loss of the c_vcl termination signal apparently resulted in overlapping convergent trans- cription of two yeast genes. This is not the case in the plasmids described here since the yeast gene closest to the 3’ end of the prochymosin gene is URA3, and both are transcribed in the same direction. Appar- ently, specific local termination within a few bases of the end of the gene is not so important for closely spaced genes transcribed in the same direction.
45
The amount of prochymosin produced in strain CGY441, about 0.5% of the TCA-extractable pro- tein (MATERMS .4m METHoDs, section c), is somewhat less than expected, considering the abun- dance of prochymosin-specific mRNA (about 7 % of the poly(A~containing RNA of strain CGY461, unpublished observations). This discrepancy could reflect inefficient translation of the prochymosin mRNA in yeast, instability of the prochymosin pro- tein, or a combination of these effects. These possi- bilities are currently under investigation. Measure- ments of the stability of prochymosin in yeast reveal a relatively long half life of about 80 min (unpublished observations). Recent work with the promoter from the yeast triose phosphate isomerase gene has result- ed in production of a prochymosin fusion protein at about 2.5% of the cell protein (equivalent to 2% of prochymosin, correcting for molecular weight of the fusion protein). The prochymosin-specific mRNA level produced from this construction is comp~able to that produced from the galactokinase promoter (unpublished observations). Thus, efficiency of translation of the mRNA appears to be a more significant factor in production of prochymosin than protein stability, and the differences in the nucleotide sequence in the translation initiation region may affect the tr~slation efficiency.
Two other observations concerning the level of prochymosin production are interesting. First, the different yeast 4 plasmid fragments present in plasmids pCGS 168 and pCGS242 apparently result in a two- to five-fold difference in the level of expression of prochymosin protein and mRNA. While both fragments contain the 2~ origin region (Broach and Hicks, 1980), the fragment in pCGS242 is slightly larger and could contain a &-acting region important for higher plasmid copy number (Jayaram et al., 1983). An increase in copy number could lead to a higher prochymosin protein yield in cells carry- ing that plasmid.
Second, the amount of prochymosin obtained from yeast depends critically on the extraction method. About 80% of the prochymosin in yeast strain CGY461 is not freely soluble in the cytoplasm after cell breakage with glass beads. The cellular location of this insoluble fraction is not currently known, but the phenomenon may be analogous to the presence of insoluble inclusion bodies in E. coli
cells synthesizing insulin chains (Williams et al.,
1982). The nature and cellular location of this pro- chymosin fraction are currently under investigation.
Production of calf prochymosm in a microorga- nism is of interest not only because it offers insights into the mechanism of heterologous gene expression, but also because of the need for an abund~t source of the enzyme. Recently two reports have described the production of calfprochymosin in E. coli (Nishi- mori et al., 1982; Emtage et al., 1983), and Mellor et al. (1983) have described the production of pro- chymosin in S. cerevisiae, an organism more compa- tible with food processing than E. co/i. We describe here the production of calf pro~h~osin in S. cerevi-
s&e by means of an efficient regulatable promoter from the yeast GAL1 gene. The yeast-synthesized prochymosin is converted to active chymosin by the same procedures known to activate prochymosin from the calf.
ACKNOWLEDGEMENTS
We thank Joya Minicucci, Susan Hayes and Dayle Holmes for preparation of the manuscript. We thank David Botstein, Gerald Fink and Gerald Vovis for helpful discussions and for critically reading the rn~us~~pt. We thank Ronald Davis for the plasmid containing the yeast GAL1 promoter, for helpful discussions and for unpublished information. We thank Robert Goltz, Robert Stearman, Margaret Duncan, Cathy Stillman, Mary Ellen Rhinehart and Jay Lillquist for contributions important to the suc- cess of this work. This investigation was supported by a contract from Dow Chemical Company.
REFERENCES
Beggs, J.D.: Transformation of yeast by a replicating hybrid plasmid. Nature 275 (1978) 104-109.
Bennetzen, J.L. and Hall, B.D.: Codon selection in yeast. J. Biol. Chem. 257 (1982) 3026-303 I.
Berk, A.J. and Sharp, P.A.: Sizing and mapping ofearly adenovi- rus mRNAs by gel electrophoresis of Sl endonuclease- digested hybrids. Cell 12 (1977) 721-732.
Botstein, D., Faico, S.C., Stewart, S.E., Brennan, M., Scherer, S., Stinchcomb, D.T., Struhl, K. and Davis, R.W.: Sterile host yeasts (SHY): a eukaryotic system of biological contain- ment for recombinant DNA experiments. Gene 8 (1979) 17-24.
46
Broach, J.R. and Hicks, J.B.: Replication and recombination
functions associated with the yeast plasmid, 2 micron circle.
Cell 21 (1980) 501-508.
Carlson, M. and Botstein, D.: Two differentially regulated
mRNAs with different 5’ ends encode secreted and intracel-
lular forms of yeast invertase. Cell 28 (1982) 145-1.54.
Derynck, R., Singh. A. and Goeddel, D.V.: Expression of human
interferon-gamma cDNA in yeast. Nucl. Acids Res. I 1 (1983)
1819-1837.
Dobson, M.J., Tuite, M.F., Roberts, N.A., Kingsman, A.J. and
Kingsman, SM.: Conservation of high efficiency promoter
sequence in S. cerevisiae. Nucl. Acids Res. 10 (1982)
