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Description  |
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This invention relates to the use of recombinant DNA technology to produce
biologically useful polypeptides in yeast, and particularly to vectors
including the gene coding for Saccharomyces cerevisiae alpha-factor useful
for production of such polypeptides.
With the onset of recombinant DNA technology, often referred to as the
field of genetic engineering, it has become possible to make recombinant
DNA molecules which may be expressed in particular microorganisms. It has
been possible for example to modify bacteria and yeast to produce various
heterologous polypeptides, i.e., polypeptides which would not be normally
produced in the host microorganism.
A primary tool of genetic engineering is a structure called a plasmid. A
plasmid is usually a circular bit of nonchromosomal
autonomously-replicating DNA. It has been found that insertions and
rearrangements of genes within the plasmid are consistent with continued
replication and growth of the host organism. Included in the information
encoded in plasmid DNA is that required to reproduce the plasmid in
daughter cells (i.e., an origin of replication) and ordinarily one or more
characteristics which permit clones of the host cell containing the
plasmid to be selectively grown.
Another key tool of genetic engineering is a process known as
transformation. By this process one can induce genetic modification of an
organism, heretofore usually a bacterium such as E. coli, by incorporating
therein fragments of DNA from other sources, such as a plasmid. The
procedure for transformation of yeast is described by Hinnen et al., Proc.
Natl. Acad. Sci. U.S.A., 75:1929 (1978) and by Beggs, Nature, 275:104
(1978).
By a process known as selection, clones of the host cell resulting from
transformation which contain the plasmid may be selectively grown. If the
transformation is to be performed by host bacteria, a plasmid may be
chosen which contains a gene for resistance to an antibiotic. If the
transformation is to be performed in yeast, a plasmid may be chosen which
contains a gene conferring the ability of cells to grow in the absence of
a required nutrient, such as, a particular amino acid. Therefore,
depending on the selection trait of the plasmid, only clones containing
the plasmid will grow in cultures containing the antibiotic or in cultures
without the required nutrient.
The utility of plasmids lies in the fact that they can be specifically
cleaved by one or another restriction endonucleases (also known as
restriction enzymes) each of which recognizes a particular sequence of
nucleotides and cleaves them in a particular way. See R. B. Wickner,
Methods of Molecular Biology, Vol. 9 (Marcel Dekker, Inc., New York);
Mertz et al., Proc. Natl. Acad. Sci. U.S.A., 69, 3370 (1972). Usually the
cleavage of double stranded DNA by a particular restriction enzyme results
in two so-called sticky ends at which the two DNA strands are of unequal
length by one to four nucleotides. These ends can only be joined (ligated)
to complementary sticky ends which have been cleaved by the same
restriction enzyme. The enzymes which join ends of fragment DNA are known
as ligases. If one needs to join a sticky end to another,
non-complementary fragment, then the sticky ends are usually enzymatically
filled in to make the DNA strands of equal length (blunting). See
Hanningson, Proc. Natl. Acad. Sci. U.S.A., 65, 268 (1970). The same
ligating enzyme used in higher concentration will join blunted fragments
(see Sgaramella et al., J. Mol. Biol., 72, 427-44 (1972)). The theory of
genetic engineering therefore is to begin with a plasmid wherein the
relative locations of various genes, including genes for selectable
characteristics, are known along with the various locations of particular
restriction sites (which are cleavable by particular enzymes) and to
cleave the plasmid with restriction enzymes. Thereafter heterologous genes
or gene fragments may be inserted into the plasmids at a cleavage site.
The recombinant plasmid may be introduced into an organism by
transformation. By various selection and purification processes the
desired transformant may be isolated and grown and which thereby produces
a desired heterologous polypeptide.
In yeast the location of the translational start and translational stop
signal on mRNA derived from a recombinant plasmid is especially important
since if they are not precisely placed to bracket the desired structural
gene, a fusion protein will result which may include only part of the
amino acid sequence of the desired end product as well as one or more
additional lengths of superfluous protein.
A further problem which may occur with use of bacteria derived plasmids to
produce mammalian proteins on an industrial scale is that bacteria do not
utilize the protein secretion and protein processing mechanisms found in
eukaryotes. Therefore, while bacteria may be used to make mammalian
proteins, the presence of heterologous proteins in the bacterial cell may
have an adverse effect on cell metabolism. However, since yeast is known
to have protein secretion and protein processing mechanisms similar to
those in other eukaryotes, it may be possible for the yeast cells to
remain healthy during production of the heterologous proteins. There has,
however, heretofore not been a method by which one could assure the
secretion of a heterologous peptide from yeast cells.
It is therefore an object of the present invention to provide a method for
causing heterologous polypeptides expressed in yeast from recombinant DNA
to be secreted from the yeast cells.
It is a further object of the present invention to provide a method for
expression of useful heterologous polypeptides in yeast which do not
contain fused superfluous amino acid sequences.
The following is a brief description of the drawings.
FIGS. 1A and 1B are the coding sequence for alpha factor precursor and the
amino acid sequence of expressed alpha factor precursor.
FIG. 2 is a partial schematic of the alpha-factor precursor illustrating
the relationship of the spacer and alpha factor segments.
FIG. 3 is an illustration of the approach described in Example 9a for
fusion of a segment of the precursor for somatostatin to an amino-terminal
segment of alpha-factor gene.
FIG. 4 is an illustration of the approved described in Example 96 for
fusion of the corticotropin (ACTH) gene to an amino terminal segment of
the alpha-factor precursor gene.
FIG. 5 is an illustration of the approach described in Example 9c for
construction of a hybrid alpha-factor precursor-enkaphalin polypeptide.
The present invention provides a method for cloning the gene for the
polypeptide alpha-factor, which is a secreted polypeptide in the yeast
Saccharomyces cerevisiae. The alpha-factor is a polypeptide composed of
twelve and thirteen amino acids which is secreted by S. cerevisiae cells
of the alpha mating type, but not by cells of the a or a/alpha types. The
fusion by known methods of the alpha-factor gene to genes of other useful
proteins such as the genes for interferon, hepatitis coat protein, etc.,
may cause the expression of a fused protein and secretion thereof from the
yeast cell into the growth medium. In addition, these fusions may result
in the production of interferon, hepatitis coat protein, etc. that lack
superfluous amino acids.
According to the present invention it has been found that the alpha-factor
precursor gene comprises a plurality of sequences coding for alpha-factor,
particularly four, separated from one another by spacer nucleotide
regions. It has been found that these spacer regions provide peptides
which are substrates for the yeast proteolytic processing system. Thus, by
deleting the alpha-factor coding sequences in the alpha-factor precursor
and by inserting other useful proteins between the spacer coding regions,
or by fusing to these regions, the resultant fusion polypeptide is not
only be expressed, but also may be secreted from the yeast cell and
processed by the yeast proteolytic processing enzymes to yield multiple
copies of the desired protein.
Mating in yeast appears to be facilitated by oligopeptide pheromones
("mating factors") that cause arrest of cells of opposite mating type in
the G1 phase of the cell division cycle. Yeast alpha cells produce the
alpha-factor, which has two forms, 13 and 12 L-amino acids in length, the
latter lacking the N-terminal Trp residue of the tridecapeptide. Yeast a
cells produce a-factor, which is 11 L-amino acids in length and has two
forms that differ in the sixth residue (Leu or Val). Synthesis of
alpha-factor requires cellular RNA and protein synthesis (Scherer et al.,
J. Bacteriol. 119:386 (1974)). Based on these observations and by analogy
with mammalian peptide hormones, there is support for the proposal that
the yeast mating factors are derived by proteolytic processing from a
larger precursor. We have thus cloned the alpha-factor structural gene
(MAF alpha, "alpha mating factor") and determined the structure of the
precursor.
The alpha-factor precursor gene was cloned as follows. A clone bank in
plasmid YEp13 carrying random segments of yeast DNA was transformed into a
mat alpha-2 strain and screened for colonies able to secrete alpha-factor
by halo formation on a lawn of a cells. One such plasmid with a 4 kb
insert (plasmid MAF alpha) was obtained. The gene responsible for halo
formation is on 1.7 kb EcoRI fragment and is inactivated by cleavage with
HindIII. Sequencing adjacent to the HindIII site showed a nucleotide
sequence of approximately 800 base pairs as shown in FIG. 1. The 165 amino
acid peptide produced from the translation of nucleotide positions 1-495
in FIG. 1 is the alpha-factor precursor.
The first 20 to 22 amino acids of the alpha-factor precursor comprise a
polypeptide segment that is structurally similar to amino terminal
segments of other proteins from bacteria and mammalian cells which are
secreted from the cells. Therefore, this 20 to 22 amino acid region is
called the signal peptide which provides the information which directs the
alpha-factor precursor and ultimately then the mature alpha-factor to be
secreted. The first half of the precursor also contains three potential
sites for glycosylation (asn-X-thr), at positions 23-25, 57-59 and 67-69,
which may also be essential for secretion.
The mature form of the alpha-factor is thirteen amino acids long and it may
be seen from FIG. 1 that the precursor codes for four tandemly arranged
copies of the mature alpha-factor. The sequence from nucleotide 250-495 is
schematically shown in FIG. 2, wherein the four alpha-factor genes are
alpha F1, alpha F2, alpha F3 and alpha F4 and the spacer sequences are S1,
S2, S3 and S4. The four alpha factors have the same amino acid sequence
but the corresponding nucleotide sequences differ by a total of 7
nucleotides. The nucleotide sequences of S3 and S4 are identical and they
code for Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala. In S2, Glu replaces Asp and S1
is shorter than the other spacer sequences by two amino acids. Therefore,
because the mature form of the alpha-factor is thirteen amino acids in
length, the precursor must be processed by proteolytic activities present
in yeast cells which cleave the mature alpha-factors from the spacers.
These spacer peptides are presumed to contain the recognition regions
which determine how the precursor is proteolytically processed. The end of
the precursor occurs immediately after the final alpha-factor coding
sequence so the processing of the precursor and secretion of the
alpha-factor does not appear to require the presence of any amino acids on
the carboxy- terminal end of the precursor peptide.
We have found that the plasmid MAF alpha carrying the alpha-factor gene
does not secrete alpha-factor when the plasmid is present in a cells, and
produces only low levels when present in yeast mutants that are defective
in the MAF alpha-1 gene. Also we observe that yeast strains carrying a
wild type MAF alpha gene overproduce alpha-factor, i.e., they secrete more
alpha-factor than do MAT alpha strains which do not carry the MAF alpha
plasmid.
The nucleotide sequence shown in FIG. 1 identifies several sites for
recognition by restriction endonucleases. These are particularly, a PstI
site at positions 23-28, a TaqI site at position 143-146, and HindIII site
at positions 263-268, 326-331,389-394, and 452-457. By use of conventional
restriction endonuclease methods other coding information may be readily
fused to the signal peptide or to other regions of the alpha-factor gene.
For example, fusion of the coding information for interferon, hepatitis
protein or other protein to the amino terminal segment of the alpha-factor
gene may result in the secretion of a fused protein from the yeast cell
into the growth medium.
Furthermore, derivatives of the alpha-factor precursor gene (MAF alpha
gene) may be constructed wherein the alpha-factor coding sequences are
deleted but which maintain the spacer peptides and their associated
HindIII sites. The insertion of other coding sequences between the spacer
peptides by conventional methods will result in the production of a
protein containing multiple copies of the inserted coding sequence. Such a
modified precursor peptide will not only be secreted, but, because of the
presence of the spacer peptide sequences, will also be processed by the
yeast proteolytic processing enzymes to yield multiple copies of the
mature, native polypeptide.
It will be readily apparent to those of ordinary skill in the art that many
conventional modifications may be made of the alpha-factor to utilize its
characteristics of causing protein secretion and yeast proteolytic
processing. For example, since the production of alpha-factor from the MAF
alpha plasmid requires a functional MAT alpha-1 gene, a plasmid
constructed of MAF alpha and a yeast mutant which produces a
temperature-sensitive MAT alpha-1 protein (P. J. Kushner et al., Proc.
Nat. Acad. Sci., U.S.A., 76; 5264-5268 (1979)), which is a strain having
functional alpha-1 activity at room temperature (25.degree. C.) and
non-functional alpha-1 activity at 34.degree. C., is one in which the
production of hybrid proteins may be controlled by controlling the growth
temperature of the culture.
The alpha factor gene may also be put under other types of control, such as
by fusion to a yeast GAL gene which may lead to an alpha factor gene which
is activated by the sugar galactose.
By fusion of coding information for polypeptides of commercial importance
to the alpha-factor gene, a high number of copies of the polypeptides may
be produced in yeast and secreted from the yeast cells and easily isolated
for further purification. The supernatant fluid from the culture medium,
for example, may be collected by filtration to separate the fluid from the
cell matter and the polypeptide may be isolated from the culture filtrate.
Because only a small fraction of all proteins produced by yeast cells is
secreted, the culture filtrate will be greatly enriched in the polypeptide
of interest. Isolation of the polypeptide of interest from the culture
fluid therefore represents a great initial purification of this material
in comparison with previous methods in which the material of interest is
not secreted, in which case it must be purified away from the many
proteins of the yeast cells.
A fused protein produced by yeast cells will have to be proteolytically
processed as a part of the industrial preparation of the polypeptide, to
separate the alpha-factor segment from the polypeptide of interest. Some
of the proteolytic processing, however, may be accomplished by the yeast
cells themselves, which would result in a secretion of the mature
polypeptide of interest. For example, fusion of the coding sequence for
the polypeptide of interest to the HindIII site at positions 263-268
leaves intact the first spacer peptide which may be the recognition signal
for proteolytic processing by yeast enzymes. In such a case the yeast cell
may secrete the mature form of the polypeptide of interest, thereby
obviating the necessity of processing a fusion product as part of the
industrial preparation of the polypeptide. In any event, another important
advantage of the present invention is that the secreted polypeptide,
whether in mature form or as a fusion product, will be outside of the cell
and not subject to proteolytic activity capable of destroying the
polypeptide within the yeast cell. Therefore another advantage of
secretion of the polypeptides or fusion product from the yeast cell is to
result in stabilization of the polypeptide.
As described hereinabove, by deletion of the coding sequences of the
alpha-factors from the precursor gene and insertion of the coding
sequences of heterologous polypeptides, a precursor peptide may be
produced containing multiple copies of the heterologous polypeptide
flanked by spacer peptides. Since the spacer peptides may provide the
proper signals for proteolytic processing by the yeast proteolytic system,
such a genetic construction may result in secretion of multiple copies of
the mature polypeptide from the yeast cells. Therefore, strains carrying
multiple copies of the heterologous polypeptide would exhibit great
overproduction of the desired peptide.
Furthermore, by constructing coding sequences using the spacer sequences,
the spacers may be placed in sequences for larger polypeptides to provide
processing sites which are valuable in several ways. For example, a
modified insulin gene may be constructed wherein the sequences that code
for the a and b chains of insulin are separated by the sequence coding for
the spacer peptide. Such a spacer-insulin hybrid protein would be
processed into a and b fragments by the yeast processing enzymes. Another
use of the spacer sequence would be to insert the spacer sequence before a
polypeptide, such as somatostatin, to form a structure as follows:
alpha-factor precursor (residues 1-83)--spacer 1--somatostatin. Processing
of this hybrid precursor by the alpha-factor processing system would yield
somatostatin with no additional amino acid residues at its amino terminus.
This use of the alpha-factor spacer would allow production of somatostatin
in yeast which is identical to natural somatostatin.
The present invention is more fully described by, but not limited to, the
following examples.
EXAMPLE 1
Isolation of Plasmid p69A which allows Production of Alpha-Factor by mat
alpha-2 Mutants
We have used a clone bank as described in Nasmyth et al., Proc. Nat. Acad.
Sci. U.S.A., 77: 2119 (1980), that contains random genomic fragments of
yeast DNA inserted into high copy number plasmid YEp13. This plasmid
contains the origin of replication of the yeast 2u plasmid and is present
in 30-50 copies per yeast cell (See Broach et al., Cell 21:501 (1980)). A
mat alpha-2 leu2 recipient (strain XK41-10b) was transformed with plasmid
DNA isolated from the clone bank, and 17,000 Leu.sup.+ transformants were
selected, pooled, and replated on selective medium (lacking leucine).
Approximately 50,000 colonies were screened for production of alpha-factor
by the halo method. One colony (K69) was found that exhibited a small,
distinct halo on lawns of a tester cells. To confirm that the halo
resulted from production of alpha-factor and not, for example, from
production of killer toxin, which also forms a halo on sensitive hosts, we
assayed K69 for alpha-factor by the confrontation assay described below.
K69 secreted a factor that elicited cell cycle arrest and aberrant cell
morphology in a cells, indicating that it produces alpha-factor.
Production of alpha-factor by K69 requires presence of a plasmid (p69A) in
these cells: colonies grown nonselectively (in the presence of leucine)
that lose the Leu.sup.+ plasmid also lose halo-forming ability. Plasmid
p69A thus appears to contain a determinant that allows the mat alpha-2
recipient to produce alpha-factor.
EXAMPLE 2
Behavior of Plasmid p69A in Different Strains
Plasmid p69A was introduced into various yeast strains differing in their
mating type locus and in the BAR1 gene to determine whether it affected
alpha-factor synthesis. These results are shown in Table 1.
Alpha-factor was assayed by halo formation on lawns of the most sensitive a
tester strain (RC631), which carries the sst2-1 mutation. As expected,
re-introduction of p69A into a mat alpha 2-4 strain yielded cells that
produced alpha-factor. This strain is still defective in mating; hence,
p69A does not contain a functional MAT alpha-2 gene. However, MAT alpha
strains carrying p69A produced a considerably larger halo than did MAT
alpha strains carrying YEp13. A further indication that p69A leads to
overproduction of alpha-factor is that wild type MAT alpha strains
carrying p69A form a halo on MATA SST2.sup.+ lawns. Plasmid p69A thus
contains a gene whose expression is limiting for alpha-factor production
in a wildtype alpha cells as well as in mat alpha-2 mutants. The
observation that (p69A)/mat alpha 1-5 strains produce very little
alpha-factor is not unexpected since alpha 1 product is necessary for
alpha-factor synthesis.
TABLE 1
______________________________________
Production of alpha-factor by strains carrying
plasmid p69A.
alpha-factor production
Nuclear assayed on tester lawns
Plasmid Genotype MATa sst2-1 MATa SST2
______________________________________
p69A mat alpha 2-4
+ -
p69A MAT alpha +++ +
p69A mat alpha 1-5
+/- -
YEp13 MAT alpha ++ -
p69A MATa bar1-1 +/-.sup.a -
p69A MATa BAR1.sup.b
- -
______________________________________
Legend to Table 1
Strains carrying various plasmids were tested for alpha-factor production
by the halo assay at 30.degree. on BBSD medium (to select for maintenance
of the plasmid) as described below. MATa testers were strains 227 (SST2)
and RC231 (sst2-1). ++, wildtype alpha-factor halo; +, halo smaller than
wildtype; +++, halo larger than wildtype; +/-, small halo; -, no halo.
.sup.a Halo size varies in different segregants from no halo to a halo as
large as by (p69A/mat alpha 1-5 strains. .sup.b This (p69A)/MATa strain
did not show an increased level of a-factor. mat alpha 2-4, mat alpha 1-5,
and MATa strains carrying YEp13 did not produce alpha-factor.
EXAMPLE 3
Sequencing of Plasmid p69A
Prior to sequencing, the determinant for alpha-factor synthesis was
localized within the 4 kbp insert present in plasmid p69A. The insert
contains three EcoRI sites, which define segments R1-1, R1-2, R1-3 and
R1-4. The R1-2 fragment contains a cluster of four HindIII sites (see FIG.
1) which are separated from each other by 63 bp. Plasmids lacking
different EcorRI fragments were constructed by partial digestion with
EcoRI followed by religation and were tested for alpha-factor production
in XK41-10b. These results indicate that the alpha-factor determinant is
located in the 1.7 kbp fragment R1-2: all plasmids containing R1-2 allow
alpha-factor production, whereas all plasmids lacking this fragment do not
(see Table 2 below). Plasmids that carry either the region of R1-2 to the
left of the HindIII site cluster (YEp13-H2) or to the right of the cluster
(YEp13-H1) do not produce alpha-factor. These results indicate that the
HindIII sites of R1-2 lie within the gene responsible for alpha-factor
production.
The sequencing strategy of fragment R1-2 is as follows. See FIG. 2. First,
HindIII sites 1 (position 268) and 4 (position 457) were 3' end-labelled,
and the sequences to the right of site 4 (and to the left of site 1) were
determined. The last T of HindIII site 4 (position 457) is the start of a
sequence that codes for the alpha-factor tridecapeptide, which is followed
immediately by a TAA translational termination signal. Sequencing
leftwards from a Sa1I site forty basepairs downstream of the alpha-factor
coding sequence revealed the presence of four tandem sequences coding for
alpha-factor. Each of these sequences is preceded by a similar coding
sequence of 18 or 24 nucleotide pairs. Further sequence determination
reveals an ATG located 89 amino acids upstream from the first alpha-factor
sequence. We have sequenced both DNA strands between the HinfI site 1 and
Sa1I, which includes the entire coding region and some flanking regions.
The remainder of the sequence has been determined for only a single DNA
strand. The sequence is shown in FIG. 1.
Nucleotide sequence analysis thus revels that plasmid p69A contains
sequences coding for alpha-factor that are contained within a large coding
sequence, an alpha-factor precursor. The key features of this sequence
are: (1) The precursor is 165 amino acids long, beginning with ATG and
ending at TAA immediately following alpha-factor sequence 4. (2) The
precursor contains four sequences coding for identical alpha-factor
tridecapeptides (alpha-factor 1, 2, 3, 4). (3) Between these alpha-factor
sequences are regions coding for similar octapeptides, which we refer to
as peptide "spacer" S2, S3, and S4. Preceding alpha-factor 1 is a similar
amino acid sequence of six amino acids (spacer 1, S1).
TABLE 2
______________________________________
Localization of the alpha-factor determinant in p69A
YEp13 derivative
alpha-factor production by
containing fragments
mat 2-4 strain carrying plasmid
______________________________________
R1-1, R1-2, R1-3, R1-4
+
R1-4 -
R1-1, R1-4 -
R1-3, R1-4 -
R1-1, R1-3, R1-4
-
R1-1, R1-2, R1-4
+
R1-2, R1-3, R1-4
+
H1 -
H2 -
none -
______________________________________
Legend to Table 2
The plasmid carrying fragment H1 contains the H1 HindIII fragment from p69A
inserted into YEp13. All other plasmids are deletion derivatives of p69A.
Plasmids were introduced into mat alpha 2-4 strain XK41-10b and tested for
alpha-factor production as described below.
EXAMPLE 4
Strains and Plasmids
Strains are given in Table 3. The yeast clone bank was constructed in
vector YEp13 by insertion of yeast genomic DNA fragments partially
digested by endonuclease Sau3a. YEp13 is a derivative of pBR322 containing
the yeast LEU2 gene and a yeast origin of replication from the 2u plasmid
(Broach et al., Gene 8:121 (1979)). Plasmid pBR322-MAT alpha (containing
the EcoRI fragment of MAT alpha) as described by Nasmyth and Tatchell Cell
19: 753-764 (1980). YEp13-MAT alpha, which contains a HindIII subfragment
from pBR322-MAT alpha, was also constructed.
TABLE 3
______________________________________
Strain List
Strain Relevant Genotype
______________________________________
DC5 MATa leu2-3 leu2-112
(Broach et al, Gene
his3 can1 8:12 (1979))
DC6 MAT leu2-3 leu2-112
G121c3-24a
his4 can1 mat 2-4 cry1
XK41-10b leu2 his4 mat 2-4 cry1
(segregant from DC5
HMLa leu2-3 leu2-112
X G121C3-24a)
RC629 MATa sst1-2 rme ade2-1
(Chan et al., Molec.
his6 Cell Biol. 2:11 (1982))
RC631 MATa sst2-1 rme ade2-1
(Chan et al., supra)
AB320 his6 HO ade2-1 lys2-1
(Nasmyth et al.,
trp5-2 leu1-12 Proc. Nat. Acad. Sci.
U.S.A., 77:2119 (1980))
G116-4A mat 1-5 cry1 ura3 trp1
his4 leu2
XK96A2-6b
mat 1-5 cry1 leu2-3
(segregant from
leu2-112 trp1 G116-4A X DC5)
G245-24C MATa bar1-1 leu2-3
leu2-112 trp1
227 MATa lys1 cry1
______________________________________
Media
E. coli were grown on LB agar supplemented as necessary with ampicillin
(100 ug/ml) or tetracycline (20 ug/ml). Yeast complete medium (YEPD) and
synthetic minimal medium (SD) are described in Hicks et al., Genetics
83:245 (1976). Alpha-factor halo assays were performed on YEPD and SD
media (BBMB and BBSD, respectively), buffered as described by Fink et al.
Proc. Nat. Acad. Sci., U.S.A., 69:2846 (1972).
Enzymes and Radiochemicals
Restriction endonucleases were obtained as follows: Fnu4H (gift from D.
Russell and M. Smith), EcoRI (Miles Laboratories), and other enzymes from
New England Biolabs. DNA ligase was obtained from P-L Biochemicals, calf
alkaline phosphatase (grade I) and E. coli DNA polymerase I (Enzym A n.
Klenow) from Boehringer Mannheim. alpha.sup.32 P-dATP, used for
end-labelling of DNA fragments, were obtained from Amersham (2000-3000
ci/mmol) or from New England Nuclear (600 Ci/mmol or 2000-3000 Ci/mmol).
EXAMPLE 5
Assays
Alpha-factor Assays: Production of alpha-factor by colonies or patches of
cells was assayed on agar medium by the halo method (modified from Fink et
al., supra) or on thin agar slabs by the confrontation assay (Duntze et
al. Science 168: 1472 (1970)). Halo assay: Approximately 10.sup.7 a cells
were spread on BBMB or BBSD plates and were imprinted by replica plating
with colonies or patches of cells to be tested for alpha-factor
production. In most assays, the a tester was strain RC631, which carries
the sst2-1 mutation (Chan et al., supra). Under these conditions, mat
alpha 1-5 and mat alpha 2-4 mutants produce a halo at room temperature but
not at 30.degree. or 34.degree..
Confrontation assay: A line of cells (approximately 10.sup.5 -10.sup.6) to
be tested for alpha-factor production was streaked on a thin agar slab
(either YEPD or minimal medium). Individual a cells (usually MATa sst1-2
strain RC629) were then placed near the line of cells by micromanipulation
and observed for response to alpha-factor (inhibition of budding,
formation of elongated cells). Transformants carrying YEp13-derived
plasmids were tested on minimal medium lacking leucine (to select for the
plasmid) and were incubated overnight at 30.degree. before introducing the
a tester cells.
EXAMPLE 6
Screening Procedures For Identification of Plasmids Carrying the MAF Alpha
Gene
Plasmid DNA was extracted from the E. coli-yeast pool and used to transform
leu2 mat alpha-2 strain XK41-10b to Leu.sup.- by selection on minimal
medium. These colonies were collected and replated on selective medium and
then assayed at 30.degree. for production of alpha-factor by the halo
assay. Optimal conditions for this assay were developed by monitoring halo
formation by colonies of strain XK41-10b carrying plasmid YEp13-MAT alpha.
Maximal halo size was obtained under the following conditions: (a)
alpha-factor assays were performed on minimal medium to select for
maintenance of YEp13. (2) The tester strains used for the alpha-factor
halo assay carried a mutation in the SST2 gene, which leads to
supersensitivity to alpha-factor (Chan et al., supra).
EXAMPLE 7
Construction of Plasmid Derivatives
The HindIII fragment containing the MAT alpha locus was transferred from
pBR322-MAT alpha to YEp13 by cleavage of pBR322-MAT alpha with HindIII and
ligating to HindIII-cleaved YEp13 which had been treated with calf
alkaline phosphatase. YEp13 carrying the 1.3 kbp HindIII fragment of p69A
(plasmid YEp13-H2) was constructed in a similar manner. A derivative of
p69A lacking the H1 fragment (plasmid YEp13-H1) was obtained by cleavage
of p69A with HindIII followed by circularization. Derivatives of p69A
lacking different EcoRI fragments were constructed by partial digestion of
p69A with EcoRI followed by ligation. Amp.sup.R E. coli transformants that
complement the bacterial leuB mutation must contain the 4.6 kb, 4.0 kb and
2.3 kb EcoRI fragments (which includes a small fragment of the yeast
insert in p69A (fragment R1-4)). Presence of additional EcoRI fragments
(R1-1, 1.1 kb; R1-2, 1.7 kb; R1-3, 1.0 kb) was determined by restriction
endonuclease digests. Plasmids were then transformed into the mat alpha
2-4 strain and tested for their ability to produce alpha-factor.
EXAMPLE 8
DNA Sequencing and Hybridization
The sequencing procedure of Maxam and Gilbert (Proc. Nat. Acad. Sci.
U.S.A., 24:560 (1977); Methods in Enzymol 65:499 (1980)) was used. DNA
fragments were labelled by the 3' end labelling technique (Smith et al.,
Cell 16:753 (1979)). All restriction endonuclease sites used for labelling
DNA ends have been read across in an independent sequence analysis.
Hybridizations to restriction endonuclease fragments were performed by the
method of Southern (J. Mol. Biol. 98:503 (1975)).
EXAMPLE 9a
Fusion of a Segment of the Precursor For Somatostatin to an Amino-Terminal
Segment of the Alpha-Factor Gene
The R1-2 fragment of the alpha factor gene is cleaved with HindIII to
produce a fragment (denoted RH1). The cohesive end of the HindIII site of
this fragment is filled in enzymatically to produce a fragment denoted RH2
to be ligated to a segment of the somatostatin gene. The RH2 fragment is
jointed to a PstI-EcoRI fragment (denoted PE) from the sequence that codes
for pre-prosomatostatin (which is described in Goodman, R. H., J. W.
Jacobs, W. W. Chin, P. K. Lund, P. C. Dee, and J. F. Habener, Proc. Natl.
Acad. Sci., U.S.A., 77:5869-5873 (1981). The DNA segment described by
Goodman et al. was produced by enzymatic conversion of pre-prosomatostatin
mRNA to double-stranded DNA by reverse transcription. Thus the
pre-prosomatostatin segment contains no intervening sequences and may be
expressed in yeast when fused to yeast expression sequence provided by the
MAF alpha gene. The RH2 segment, containing the first part of the
alpha-factor gene, is fused with the PE fragment from pre-prosomatostatin
by blunt end ligation to yield a structure shown in FIG. 3. This segment
is inserted into a high copy number yeast plasmid cloning vector and
introduced into yeast by transformation. Since the hybrid alpha
factor-somatostatin gene contains the information for secretion of yeast
polypeptides (contributed by the alpha-factor segment), the fused protein
will be secreted from yeast cells. Alternatively, yeast processing
activities that are known to process the alpha factor precursor
polypeptide will cleave after the lysine residue (at position 105 in the
somatostatin segment) to liberate native somatostatin. No processing at
the carboxy terminal end of somatostatin is necessary because the
somatostatin coding sequence is followed by a translation termination
signal.
EXAMPLE 9b
Fusion of a Segment of the Corticotropin (ACTH) Gene to an Amino Terminal
Segment of the Alpha Factor Precursor Gene
The RH2 fragment described in Example 9a is joined to a DNA segment
containing coding information for ACTH, such as, the sequence coding for
bovine pre-pro ACTH. The bovine ACTH precursor segment is described by S.
Nakanishi, A. Inoue, T. Kita, M. Nakamura, A. C. Y. Chang, S. N. Cohen,
and S. Numa, Nature 278: 423-427 (1979). The segment is a double-stranded
DNA derivative of the corresponding mRNA and thus does not contain any
intervening sequences. This segment contains sites for the restriction
endonuclease SmaI which cuts between amino acid residues -16/-15 and
between residues 86/87 to yield a DNA segment denoted SS. ACTH (positions
1-39) is located between these SmaI sites. The RH2 fragment from the alpha
factor gene is joined by blunt end ligation to the SS fragment containing
ACTH (See FIG. 4). This segment is inserted into a high copy number yeast
plasmid cloning vector and introduced into yeast by transformation.
Rationale: As in Example 9a, a hybrid protein is produced and is secreted
from yeast cells. Furthermore, yeast proteolytic processing activities
cleave this hybrid precursor molecule at the Lysine and Arginine residues
that flank ACTH to liberate native ACTH, which is secreted from yeast
cells.
EXAMPLE 9c
Construction of a Hybrid Alpha Factor Precursor-Enkephalin Polypeptide
which Produces Secreted and Mature Enkephalins
The segment RH2 is described in examples 9a and 9b. A DNA segment (denoted
ME) coding for met-enkephalin (Tyr-Gly-Gly-Phe-Met) is synthesized
enzymatically according to methods described and utilized by Comb, M., E.
Herbert, and R. Crea, Proc. Natl. Acad. Sci., U.S.A., 79:360-364 (1982).
DNA segments that code for Lys-Arg and for Lys-Arg-Glu-Ala-Glu-Ala-Glu-Ala
are synthesized by analogous methods. These segments provide processing
sites (denoted PS1 and PS2, respectively). Met-enkephalin segments are
jointed to PS1 or to PS2 segments by blunt end ligation. The (ME, PS1) and
(ME, PS2) ligation mixtures are joined to RH2 by blunt end ligation and
are inserted into a high copy number yeast plasmid cloning vector (see
FIG. 5). The population of resultant plasmids is introduced into yeast
cells and assayed for secretion of enkephalins. The yeast alpha-factor
segment provides coding signals for secretion of the precursor molecule.
The mature enkephalin molecules are cleaved from the hybrid precursor by
yeast proteolytic activities that cleave after Lys-Arg or after
Lys-Arg-Glu-Ala-Glu-Ala-Glu-Ala. Lys residues are removed from the carboxy
terminus of enkephalin by yeast carboxypeptidase.
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