U.S. patent application number 13/536580 was filed with the patent office on 2013-03-07 for production of recombinant selenoprotein mutants with enhanced catalytic activity.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION. The applicant listed for this patent is Hangsik Moon, Richard Sayre. Invention is credited to Hangsik Moon, Richard Sayre.
Application Number | 20130059334 13/536580 |
Document ID | / |
Family ID | 37595783 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130059334 |
Kind Code |
A1 |
Sayre; Richard ; et
al. |
March 7, 2013 |
PRODUCTION OF RECOMBINANT SELENOPROTEIN MUTANTS WITH ENHANCED
CATALYTIC ACTIVITY
Abstract
The present invention generally relates to the production of
industrially relevant quantities of selenoprotein enzymes in
eukaryotic cell cultures. More specifically, the present invention
generally relates to the production of such enzymes wherein one or
more catalytic cysteine or serine residues are mutagenically
replaced by selenocysteine.
Inventors: |
Sayre; Richard; (Los Alamos,
NM) ; Moon; Hangsik; (Morrisville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sayre; Richard
Moon; Hangsik |
Los Alamos
Morrisville |
NM
NC |
US
US |
|
|
Assignee: |
THE OHIO STATE UNIVERSITY RESEARCH
FOUNDATION
Columbus
OH
|
Family ID: |
37595783 |
Appl. No.: |
13/536580 |
Filed: |
June 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11922707 |
Mar 11, 2010 |
8236936 |
|
|
PCT/US06/24093 |
Jun 21, 2006 |
|
|
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13536580 |
|
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60692875 |
Jun 21, 2005 |
|
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Current U.S.
Class: |
435/69.1 ;
435/212; 435/471; 435/472; 435/476 |
Current CPC
Class: |
C12Y 304/22002 20130101;
A61K 38/00 20130101; C12N 9/1029 20130101; C12N 15/8257 20130101;
C12N 9/00 20130101; C12P 21/02 20130101; C12N 9/6472 20130101; C12N
15/8216 20130101; C07K 14/415 20130101; C12N 15/8238 20130101; C12N
15/8218 20130101; C12N 15/8251 20130101 |
Class at
Publication: |
435/69.1 ;
435/212; 435/471; 435/476; 435/472 |
International
Class: |
C12N 9/48 20060101
C12N009/48; C12N 15/87 20060101 C12N015/87; C12P 21/00 20060101
C12P021/00; C12N 15/79 20060101 C12N015/79 |
Claims
1-8. (canceled)
9. A mutant papain enzyme, comprising selenocysteine.
10. The mutant papain enzyme of claim 9, wherein cysteine-25 in the
active site is replaced by selenocysteine.
11. The mutant papain enzyme of claim 10, which exhibits enhanced
catalytic activity compared to that of papain containing
cysteine-25 in the active site.
12. A method of making a mutant papain containing selenocysteine at
amino acid position 25 located in the active site, in a eukaryotic
algal host cell, comprising: a) introducing into said eukaryotic
algal host cell an expression cassette comprising, operably linked
for expression in said eukaryotic algal host cell in the following
order: i) a tightly regulated gene promoter active in a eukaryotic
alga; ii) a nucleic acid sequence encoding papain, wherein UGA
replaces the codon encoding cysteine at amino acid position 25,
located in the active site of said papain; and iii) a 3'
untranslated eukaryotic selenocysteine insertion sequence; b)
selecting transformed cells; c) culturing selected cells in a
medium that promotes expression of said mutant
selenocysteine-containing papain; and d) recovering said mutant
selenocysteine-containing papain from said eukaryotic algal host
cell or said medium when said papain is secreted.
13. The method of claim 12, wherein said tightly regulated gene
promoter is the Fea1 promoter.
14. The method of claim 12, wherein said nucleic acid sequence
encoding papain comprises a nucleic acid sequence encoding a FLAG
tag.
15. The method of claim 12, wherein said expression cassette
further comprises a selection sequence.
16. The method of claim 15, wherein said selection sequence is an
antibiotic or auxotrophic selection sequence.
17. The method of claim 12, wherein said expression cassette
further comprises a signal sequence that causes secretion of said
papain from said eukaryotic alga.
18. The method of claim 17, wherein said signal sequence is a 5'
perplasmic targeting sequence.
19. The method of claim 18, wherein said signal sequence is that of
the Fea1 gene, which causes said papain to accumulate between the
cell membrane and cell wall.
20. The method of claim 12, wherein said expression cassette
further comprises an immuno-tagging sequence located downstream
from said signal sequence, at the N-terminal end of said
papain.
21. The method of claim 20, wherein said immuno-tagging sequence is
a FLAG epitope.
22. The method of claim 12, wherein said expression cassette
further comprises a sequence coding for an RNAi that down-regulates
expression of a gene product that interferes with expression of
said papain.
23. The method of claim 22, wherein said sequence coding for an
RNAi that down-regulates expression of a gene product that
interferes with expression of said papain is a copy of at least a
portion of a nucleic acid sequence encoding a translated wild-type
gene in reverse orientation.
24. The method of claim 23, wherein said gene product that
interferes with expression of said papain is wild-type Fea1
protein.
25. The method of claim 12, wherein said 3' untranslated eukaryotic
selenocysteine insertion sequence is Chlamydomonas selenocysteine
insertion sequence SeIKI.
26. The method of claim 25, wherein said SeIKI sequence is part of
a SeIKI terminator sequence.
27. The method of claim 12, wherein said expression cassette is
contained in an expression vector.
28. The method of claim 27, wherein said expression vector is a
plasmid.
29. The method of claim 28, wherein said plasmid is pUC18 or
pUC19.
30. The method of claim 12, wherein said expression cassette is
introduced into said eukaryotic algal host cell via a virus,
electroporation, transfection, or the glass bead method.
31. The method of claim 12, further comprising introducing into
said eukaryotic algal host cell a cassette that reduces expression
of periplasmic-targeted proteins to facilitate purification of said
selenocysteine-containing papain.
32. The method of claim 12, further comprising separately
introducing into said eukaryotic algal host cell a selection
sequence by co-transforming said eukaryotic algal host cell with an
additional plasmid comprising said selection sequence.
33. The method of claim 32, wherein said selection sequence is an
antibiotic or auxotrophic selection sequence.
34. The method of claim 12, wherein said medium contains 100
.mu.g/L sodium selenite.
35. The method of claim 12, wherein expression of expression of
said mutant selenocysteine-containing papain is induced by elevated
carbon dioxide, low iron, or high cadmium levels.
36. The method of claim 12, wherein said eukaryotic algal host cell
is Chlamydomonas.
37. A method of making a mutant protein containing selenocysteine
in a eukaryotic algal host cell, comprising: a) introducing into
said eukaryotic algal host cell an expression cassette comprising,
operably linked for expression in said eukaryotic algal host cell
in the following order: i) a tightly regulated gene promoter active
in a eukaryotic alga; ii) a nucleic acid sequence encoding said
protein, wherein UGA replaces a codon encoding cysteine at a
desired amino acid position in said protein; and iii) a 3'
untranslated eukaryotic selenocysteine insertion sequence; b)
selecting transformed cells; c) culturing selected cells in a
medium that promotes expression of said mutant
selenocysteine-containing protein; and d) recovering said mutant
selenocysteine-containing protein from said eukaryotic algal host
cell or said medium when said protein is secreted.
Description
[0001] This application is a continuation of, and claims the
benefit of priority of, U.S. patent application Ser. No.
11/922,707, filed Mar. 11, 2010, which is the U.S. national stage
filing under 35 U.S.C. .sctn.371 of PCT/US2006/024093, filed Jun.
21, 2006, which claims the benefit of priority of U.S. Provisional
Application Ser. No. 60/692,875, filed Jun. 21, 2005. The contents
of each of these prior applications is herein incorporated by
reference in their entirety.
SEQUENCE LISTING
[0002] In accordance with 37 C.F.R. .sctn.1.821(e), in lieu of
filing a duplicate computer readable form of the Sequence Listing
filed in patent application Ser. No. 11/922,707, submitted on Dec.
21, 2007, Applicants state that the computer readable form of the
Sequence Listing in this continuation application is to be
identical with the computer readable form of the Sequence Listing
submitted in parent application Ser. No. 11/922,707, which was
compliant with all of the requirements of 37 C.F.R.
.sctn..sctn.1.821-1.825.
BACKGROUND OF THE INVENTION
[0003] The present invention is generally directed to a method for
producing industrially relevant quantities of catalytically
enhanced recombinant enzymes. More particularly, the present
invention is generally directed to such a process wherein the gene
encoding the enzyme is a mutant that enables a catalytic cysteine
or serine to be substituted with the selenium analog, i.e.
selenocysteine.
[0004] Industrially relevant production of selenoproteins in
non-animal or single celled systems has proven challenging because
eukaryotic and bacterial selenocysteine insertion sequences (SECIS)
operate differently. Bacterial SECISs are usually located
immediately downstream of the UGA codon encoding selenocysteine.
Thus, bacterial SECISs are translated. In contrast, typical
eukaryotic SECISs are part of the 3' untranslated region (UTR) of
the mRNA. The upshot of this architectural difference between
bacterial and eukaryotic SECISs is that in order to express a
eukaryotic gene in a bacterial cell you must add condons to the
gene, which results in adding amino acids to the gene product.
Accordingly, expressing a eukaryotic gene in bacteria is certain to
affect the structure and likely to deleteriously affect the
function of the recombinant protein.
[0005] Previously, others have demonstrated that Chlamydomonas has
a eukaryotic SECIS. This suggested that it is possible to produce
selenocysteine proteins in Chlamydomonas. However, the prior work
was silent as to the catalytic benefit conferred by artificially
replacing wild-type cysteine with selenocysteine and methods for
secreting recombinant selenocysteine-containing proteins from
Chlamydomonas under the control of a tightly regulated gene
promoter.
[0006] The present invention provides methods for replacing
cysteine and/or serine with selenocysteine. More particularly, the
present invention provides a method of producing recombinant
selenocysteine-containing proteins using any gene source,
prokaryotic or eukaryotic, wherein the protein is catalytically
enhanced through replacing cysteine and/or serine with
selenocysteine. Accordingly, the present invention is novel and
nonobvious, and thus deserves broad patent protection.
SUMMARY OF THE INVENTION
[0007] The present invention generally relates to a method for
producing industrially relevant quantities of recombinant
selenoproteins. More specifically, the present invention relates to
a method for producing recombinant selenoprotein enzymes in which
catalytically active cysteine or serine residue(s) are replaced by
selenocysteine residues thereby yielding enzymes with enhanced
catalytic activities. The present invention further relates to
nucleic acids and cells transformed thereby that are instrumental
in the method of the present invention. The present invention also
relates to a cassette that reduces expression of other
periplasmic-targeted proteins to facilitate purification of the
secreted selenoproteins.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 is a diagram of the recombinant selenocysteine
expression vector, pMOON3;
[0009] FIG. 2 is a coomasie blue stained gel of secreted proteins
isolated from Fea1-FWT and Fea1-Sec 188 cells. Each lane has 20
.mu.g protein loaded;
[0010] FIG. 3 is a western blot of supernatant proteins isolated
from wild-type, untransformed cells (lane 1), Fea1-Sec188 (lane 2)
and Fea1-FWT (lane 3) transgenic cells. Each lane contains 2.5
.mu.g of total protein; and
[0011] FIG. 4 is a diagram of the pUC19 plasmid.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention is generally directed to a method for
producing industrially relevant quantities of recombinant
selenoprotein enzymes. More particularly, the present invention
generally relates to a method for producing recombinant enzymes
having enhanced catalytic activity due to the substitution of at
least one cysteine (Cys) or serine (Ser) residue with at least one
selenocysteine (Sec). The present invention further relates to
nucleic acids and cells transformed thereby that are instrumental
in the method of the present invention. The present invention also
relates to a cassette that down-regulates expression of other
periplasmic-targeted proteins to facilitate purification of the
recombinant selenoproteins.
[0013] The process of the present invention generally comprises the
following steps. A host is selected based upon its capacity for
producing selenoprotein in cell culture. An expression vector, such
as a plasmid, is provided for encoding the desired gene product.
The host cell is then transformed by the expression vector through
any of a variety of known methods including transfection,
electroporation, the glass bead method, and the like. Next the
cells are subjected to a selection process, which tends to kill
non-transformed cells while permitting transformed cells to
reproduce. Then the selected cells are cultured in a medium
permitting expression of the gene product. Finally, the gene
product is isolated by any of a variety of suitable processes.
[0014] In general, any of a variety of suitable host cells may be
used for producing recombinant selenoproteins. A key feature of a
suitable host includes the capacity to biosynthetically produce one
or more selenoproteins using an untranslated insertion sequence.
Both animal and eukaryotic algal cells are generally capable of
producing selenoproteins in this way. Accordingly, an embodiment of
the present invention comprises using animals or animal cell
cultures to produce recombinant selenoproteins. More preferably, an
embodiment of the present invention comprises using a eukaryotic
algal cell culture to produce recombinant selenoproteins. Still
more preferably an embodiment of the present invention comprises
using Chlamydomonas reinhardtii cell culture to produce recombinant
selenoproteins.
[0015] Any of a variety of suitable expression cassettes can
comprise an embodiment of the present invention. A suitable
expression cassette typically contains a promoter sequence, a gene
product encoding sequence, and an optional selection sequence;
although, selection sequences may be introduced separate from the
expression vector by co-transformation using an additional plasmid.
Furthermore, a suitable expression cassette may optionally contain
a signal sequence. Additionally, a suitable expression cassette may
optionally contain a sequence coding for an RNAi that
down-regulates expression of interfering genes. Expression
cassettes are generally contained within expression vectors.
[0016] The function of the control sequence is to switch expression
on and off. The promoter's switching function is ideally absolutely
on or absolutely off; however, in practice such promoters are very
unusual. Thus, a preferable promoter remains in predominantly two
states; namely, substantially on, or substantially off. More
preferably such a promoter is additionally characterized by the
capacity to induce a high degree of transcription inasmuch as this
increases downstream protein synthesis. Thus, in one embodiment the
control sequence comprises the Fea1 promoter sequence (SEQ ID
1).
[0017] The expression cassette's gene product encoding sequence can
encode any of a wide range of selenoprotein genes. Moreover,
substantially similar expression cassettes may be used to
biosynthesize any of a variety of selenoproteins by changing only
the gene product encoding sequence. Thus, the process of the
present invention may be used to produce a wide range of
recombinant selenoproteins with relatively little effort.
[0018] As noted above, the expression cassette may optionally
contain a selection sequence and/or a signal sequence. Selection
sequences are discussed in more detail below; however, in general
terms a selection sequence enables one to purge non-transformed
cells from a cell culture thus leaving substantially only
transformed cells. Signal sequences include translated portions of
a gene that cause the gene product to include a proteinaceous
targeting sequence so that it tends to accumulate in a particular
region.
[0019] Additionally, as noted above, the expression cassette may
optionally contain a sequence coding for an RNAi that
down-regulates expression of an interfering gene product. For
instance, when the Fea1 gene promoter is used in combination with a
recombinant gene product sequence the wild-type Fea1 protein may
interfere with and/or contaminate the recombinant gene product.
Thus, in such instances it is desirable to down-regulate expression
of the wild-type Fea1 gene product. Down-regulation can be
accomplished by including a copy of at least a portion of the
translated wild-type gene in reverse orientation thus forming a
fold-back structure that can be processed by the dicer complex
yielding small interfering RNA or RNAi fragments. These fragments
bind to the translated region of the wild-type gene in an antisense
orientation thereby down-regulating expression.
[0020] In practice, the expression cassette is typically
incorporated into an expression vector such as a plasmid. Any of a
variety of plasmids may be used in connection with the process of
the present invention. Characteristics of a suitable plasmid
include the ability to transform a cell. More particularly, such
characteristics include the capacity to induce one or more cells to
produce a gene product encoded on the plasmid. Preferably, such a
plasmid has the capacity to produce a gene product by a process
that can be controlled, for instance, by altering conditions such
as chemical content of the growth media. Plasmids within the scope
of the present invention include without limitation the pUC18
plasmid, and the pUC19 plasmid as shown in FIG. 4.
[0021] A plasmid containing an expression cassette can be
introduced to a host cell in any one of a variety of suitable ways,
thereby transforming the cell. For instance, in one embodiment the
host cells are transfected by a virus containing the plasmid.
Alternatively, the plasmid can be introduced to the host cell
through electroporation. A still further embodiment comprises
introducing the plasmid to the host cell through the glass bead
method. One of skill in the art will recognize that other methods
for accomplishing transformation are also suitable, and are thus
encompassed by the present invention. Any combination of the
foregoing transformation methods are also within the scope of the
present invention.
[0022] Following transformation, the cells are generally subjected
to a selection process. A wide variety of known selection methods
are consistent with the present invention. For example, an
embodiment of the present invention comprises transforming at least
one cell with a gene that confers antibiotic resistance. Thus,
transformed cells can be selected by subjecting the entire
population to antibiotics. Alternatively, an embodiment comprises
transforming a host cell with a gene that confers it with the
ability to biosynthesize one or more amino acids, such as arginine,
which it would otherwise be unable to produce on its own. Thus,
transformed cells can be selected by growing the cells on media
deficient in that amino acid. One of skill in the art appreciates
that a large number of alternative selection processes are known
that are consistent with, and are thus encompassed by, the present
invention. Any one of the foregoing selection methods explicitly
enumerated or implicitly encompassed by the present invention, or
any combination thereof, can comprise an embodiment of the present
invention.
[0023] After the cells have been subjected to a selection process
they are cultured in or on a medium that promotes expression of the
desired gene product. Any of a wide variety of known culturing
methods can comprise an embodiment of the present invention. In
general, suitable culture methods are characterized by inducing the
promoter gene to begin producing the desired gene product. For
instance, the media can contain one or more chemical species at a
sufficient level to induce translation. Conversely, the media can
be deficient in one or more chemical species such that translation
is induced. Suitable media include without limitation liquid broths
and gelatinous media. Specific growth conditions and nutrients are
limited only by the requirements of the selected host cell.
[0024] If an optional signal sequence is used then the gene product
may accumulate any of a variety of convenient locations. For
instance, a signal sequence could cause the gene product to be
secreted from the host cell. One non-limiting example of a signal
sequence is that of the Fea1 gene. The Fea1 gene codes for an iron
assimilating protein known as the Fea1 protein, and the Fea1 signal
sequence codes for a gene product that causes the Fea1 protein to
accumulate between the cell membrane and wall. Thus, host cells
lacking a cell wall secrete the enzyme rather than accumulate it.
It is appreciated by those skilled in the art that there are
numerous alternative methods for targeting gene products. Thus, the
foregoing non-limiting example is merely illustrative, and does not
exclude such alternative embodiments.
[0025] Finally, the gene product is collected or isolated by any of
a wide variety of suitable known methods. For instance, the gene
product may be precipitated, extracted, filtered, centrifuged, or
any combination thereof. More specifically, if the gene product is
secreted then the cells can be separated from the media by
centrifugation, and the gene product can be salted out of the media
and collected by filtration. Alternatively, the cells can be lysed
and then separated by centrifugation followed by salting the gene
product out of the media. A still further example comprises either
of the foregoing methods in combination with any of a variety of
suitable forms of liquid chromatography including size exclusion
chromatography, HPLC, reverse phase HPLC, and the like.
[0026] The method of the present invention encompasses a wide
variety of catalytically active proteins. More specifically, the
scope of the present invention encompasses any protein enzyme
having a Cys or Ser in its active site because such enzymes can
benefit by replacing the catalytic Cys or Ser with selenocysteine.
More specifically, the sulfhydryl of Cys and the hydroxyl of Ser
are much weaker nucleophiles than the selenium hydride of
selenocysteine, which imparts less biologically favorable
ionization and reducing potentials in comparison to selenocysteine.
Thus, replacing catalytic Cys and/or Ser with selenocysteine can
result in recombinant selenoproteins with higher catalytic turnover
numbers and improved physiological activity profiles in comparison
to the natural enzyme. Accordingly, examples of enzymes encompassed
by the process of the present invention include without limitation
hydrogenase, glycine reductase, selenoprotein B, proline reductase,
formate dehydrogenase, glutathione peroxidase, thioredoxin
reductase, selenoproteins W and P, and phospholipid hydroperoxide
glutathione peroxidase, urease, fatty acid elongase, papain, Fea1
and the like.
Example 1
[0027] In one embodiment of the present invention the recombinant
gene product comprises a selenocysteine analog of the Fea1 protein.
As shown in FIG. 1, the expression cassette of this embodiment
includes a Fea1 promoter at the 3' end (see SEQ ID 1), a gene
encoding either a wild-type or seleno-analog of Fea1 (see SEQ ID
2). The Fea1 gene includes a 5' periplasmic targeting sequence. An
optional immuno-tagging sequence known as the FLAG epitope is
located just downstream from the signal sequence (see SEQ ID 4 and
5). Finally, an untranslated Sec insertion sequence, which is part
of the SeIK1 terminator sequence, is located at the 3' end of the
expression vector (see SEQ ID 3). The gene encoding Fea1 may be
either wild-type (Fea1-FWT) or the selenocysteine analog
(Fea1-Sec188). Each of the foregoing sequence are incorporated into
multicloning site of the pUC19 plasmid expression vector (See FIG.
4 and SEQ ID 6).
[0028] In the Fea1 embodiment the expression vector's promoter
sequence is also the promoter of the Fea1 protein found in
non-transformed Chlamydomonas cells. Therefore, in order to
demonstrate that transformation was successful an optional tag
sequence was included in a translated portion of the expression
vector, thereby immuno-labeling the N-terminus of the mature
recombinant gene product. Accordingly, the expression vector's
product can be readily distinguished from that of non-transformed
cells by western blot analysis, as shown in FIG. 3.
[0029] The Western blot of FIG. 3 contains four lanes. Lane 1
contains protein isolated from wild-type, untransformed cells. Lane
2 contains protein isolated from Fea1-Sec188 transgenic cells. And
lane 3 contains protein isolated from Fea1-FWT transgenic cells.
The lane labeled "std" contains a molecular weight standard. More
particularly, lane 3 includes a FLAG-tagged version of the
wild-type Fea1 gene (Fea1-FWT), and lane 2 includes a FLAG-tagged
Fea1 gene in which a stop or Sec codon (UGA) is introduced at codon
188 (Cys, UGC). In this blot, the FLAG-epitope is introduced into
both the Fea1-FWT as well as Fea1-Sec188 genes to distinguish the
recombinant proteins from that of the non-transformed cells.
Notably, the tag sequence is not required for production purposes,
and is only used here to demonstrate successful transformation and
expression of recombinant proteins. Thus, the tag is optional,
rather than a necessary component of the present invention.
[0030] As shown in FIG. 1, the Fea1-FWT and Fea1-Sec188 genes are
cloned into the selenocysteine expression vector, pMOON3. Thus the
pMOON3 expression vector comprises either the wild-type gene or the
selenocysteine mutant gene. The pMOON3 vector further includes a
Fea1 promoter sequence that is inducible by elevated carbon
dioxide, low iron, or high cadmium levels. The pMOON3 vector also
includes a multicloning site, and a Chlamydomonas selenocysteine
insertion sequence (SECIS) denoted SeIK1 located in the 3'
position.
[0031] Both the wild-type and selenocysteine mutant pMOON3-Fea1
vectors are used in combination with the p389 plasmid to
simultaneously transform host cells. That is, cells are subjected
to either a mixture of pMOON3-Fea1-FWT and p389, or
pMOON3-Fea1-Sec188 and p389; however, transformed cells do not
contain both the pMOON3-Fea1-FWT and pMOON3-Fea1-Sec188 expression
vectors. The function of the p389 plasmid is to encode the ARG7
gene, which imparts the host cell with the capacity to produce its
own arginine. Since the host cell is an arginine auxotroph, it is
not capable of producing its own arginine in the absence of p389.
Thus, transformed cells can be selected by growing the population
on arginine deficient media.
[0032] The Chlamydomonas host, CC-424, is transformed by the glass
bead method. Transformants containing the integrated pMOON3
expression vectors and p389 selection sequence are identified by
PCR using primer sets to amplify across Fea1/SeIK1 3' UTR junction.
PCR-confirmed Fea1-FWT and Fea1-Sec188 transformants, as well as
untransformed wild-type cells (CC-424), are inoculated into 25 mL
of arginine deficient Tris-acetate-phosphate (TAP) medium. Thus,
substantially only cells that are successfully transformed are able
to survive. Following ten days of growth the cells are collected,
washed twice with TAP iron-deficient medium containing 100 .mu.g/L
sodium selenite, and inoculated into 1 liter of TAP containing
sodium selenite (100 .mu.g/L) and low iron (0.5 .mu.M Fe) thus
inducing Fea1 gene expression. Most of the gene product is secreted
in the first six hours following induction, i.e. subjection to low
iron or high carbon dioxide conditions. After four days of growth,
the cultures are adjusted to 0.1 M NaCl, 1 mM
.epsilon.-amino-n-caproic acid, and agitated at 150 rpm for ten
minutes before the cells are removed from the growth medium by
centrifugation at 4,000 g and 4.degree. C. for five minutes. The
supernatants are filtered through 0.2 .mu.m membrane to remove any
remaining cells. Solid ammonium sulfate is added to 80% saturation
and the solution is stirred for an hour at 4.degree. C. to
precipitate secreted proteins. The precipitated supernatant
proteins are collected by centrifugation at 23,400 g and 4.degree.
C. for 90 minutes, resuspended in 5 mL of buffer A (50 mM Tris-HCl,
10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
.epsilon.-amino-n-caproic acid), and reprecipitated by adding 100%
ice cold trichloroacetic acid (10% (w/v) final concentration) and
allowing the precipitate to stand for one hour at 4.degree. C. The
precipitated proteins are collected by centrifugation at 20,000 g
and 4.degree. C. for 30 minutes, and resuspended in buffer B (0.1 M
Na.sub.2CO.sub.3, 0.1 M dithiothreitol). Equal amounts of proteins
are resolved on a 12% polyacrylamide gel and visualized by staining
with Coomassie blue.
[0033] As shown below in FIG. 2, a doublet band is observed
migrating at approximately 43 kD in both the Fea1-FWT and
Fea1-Sec188 transgenic lanes. Previous results have demonstrated
that the Fea1 protein runs as single bands at 43 kD (upper arrow)
and at 33 kD (lower arrow) on SDS-PAGE. Significantly, there is no
truncated Fea1 protein (188 versus 327 amino acid full length)
present in the supernatant fractions obtained from Fea1-Sec188
cells. This indicates that a selenocysteine was successfully
introduced at the mutant stop codon thus allowing full-length
expression of the Fea1-Sec188 protein. Notably, due to limited Fea1
protein production from non-transgenic or WT cells no coomassie
blue-stained proteins are visualized in FIG. 2 indicating that no
such protein is isolated from the supernatant of untransformed or
WT cells.
[0034] Since both recombinant Fea1 genes introduced into the pMOON3
expression vector include FLAG-epitopes recombinant Fea1 proteins
can be distinguished from and that of the non-transformed cells by
western blot analysis using an anti-FLAG antibody. As shown in FIG.
3 below, two co-migrating protein bands are detected by anti-FLAG
antibody at 43 and 33 kD in the supernatant fraction obtained from
the recombinant Fea1-FWT and Fea1-Sec188 cultures. A very weak
non-specific band migrating between the 43 kD and 33 kD bands is
detected in the supernatant fraction from untransformed cells.
[0035] Thus, both the 43 and 33 kD proteins detected in recombinant
supernatant fractions (i.e. that of Fea1-Fwt and Fea1-Sec188) are
full-length FLAG-tagged Fea1 proteins. The faster migrating form
(33 kD) of the Flag-immuno-detected protein is likely either a
non-glycosylated or a partially degraded form of the Fea1
protein.
[0036] Notably, the process of the present invention in accordance
with the foregoing example produces 2 mg of Fea1-Sec188 protein per
liter of culture. Such a yield suggests that it is feasible to
produce recombinant selenoprotein mutants from algal cell cultures
in commercially relevant quantities. And, unlike bacterial cell
cultures, this includes post-translationally modified proteins.
Example 2
[0037] Another embodiment of the present invention is similar to
Example 1 except that the gene product encoding sequence codes for
a mutant papain enzyme. More particularly, Cys-25 is replaced by
Sec-25. Papain is a widely used industrial enzyme that is limited
in its application in part due to its low specific activity
relative to other proteases such as trypsin. However, replacement
of the active site's Cys-25 with Sec-25 results in enhanced
catalytic activity that increases papain's cost-competitiveness
with trypsin.
[0038] According to the papain embodiment, a selenoprotein
expression vector is provided for expressing recombinant
selenoproteins in Chlamydomonas. The vector is a plasmid
substantially similar to that of the Fea1 example above; however,
the vector codes for papain rather than Fea1. More specifically,
the plasmid comprises: 1) the highly inducible Chlamydomonas Fea1
promoter, 2) an optional Fea1 signal sequence causing secretion of
the selenoprotein gene product into the media, 3) a sequence
encoding the papain enzyme; and 4) a 3' SeIk1 Chlamydomonas
Sec-insertion sequence (SECIS) for inserting the tRNAsec into the
ribosome at engineered Sec codon(s).
Example 3
[0039] A still further example of the present invention is
essentially identical to Example 2 except that the expression
vector additionally includes a sequence encoding an RNAi for
down-regulating wild-type Fea1 protein production.
Example 4
[0040] Still another example of the present invention is
essentially identical to Example 1 except that the gene product
encoding sequence codes for a fatty acid elongase, and lacks a
signal sequence. Thus the gene product is not secreted from the
cell, but rather accumulates therein. More specifically, a plasmid
consistent with this example comprises: 1) the highly inducible
Chlamydomonas Fea1 promoter, 2) a sequence encoding the fatty acid
elongase; and 3) a 3' SaIk1 Chlamydomonas Sec-insertion sequence
(SECIS) for inserting the tRNAsec into the ribosome at engineered
Sec codon(s).
[0041] The foregoing examples are considered only illustrative of
the principles of the invention rather than an exclusive list of
embodiments. Further, since numerous modifications and changes will
readily occur to those skilled in the art, the invention is not
intended to be limited to the exact construction and operation
shown and described, and accordingly, all suitable modifications
and equivalents are within the scope of the present invention.
Sequence CWU 1
1
611203DNAArtificialvector 1taccaggaca gagtgcgtgt ggccagggca
caggcgccca tccagcagct cgccgtctaa 60gtaggccgtc catgcagtgc cggtcgggtc
cggaaccacg aaccagtggt gagggaaaac 120atcgttacgc tctgggtgag
cactacacga tgggtattcc tcaattagtt ccgggtaagc 180gacaaccgag
cgagtcgccg cgagtgcaag cagtgcaatt gacaggctga acgcggccat
240cggcaatccg cagcggaact gtctcaattt acttcgtgac ctatgtatgt
tgaatatgct 300gtcgggtcga ccagcggcca gtaggagtgg ccactcggtg
tggaagagtg ggccgcgctg 360gactgctggc gcgacctttg aacgcggaca
acttgcaaaa gtatttgatt atcatcaacg 420caaaagtgat gctggcgaat
tggagggggc gccgcgaggc acgcgccagg ctgctgcgcg 480cttgccatgc
gcgtgccggg tctgtccgag agtcgagcca agtcgctgct ttatgacaca
540acaatatatc gttagttgct ctgaaggcga ccaagaacct cgcggggcgt
gctaatgtag 600gagaaacaag caagcaaccg acacgaacca gcttgctttc
ccgccgtgca gttaatgcat 660gtgcgcatgg atgcatgaaa ttcctatgga
agctgcgcat ttcccacatt gaaaaacgag 720cgcgaaaaac gcgcgtagga
gtgcatcgtg cgtgcctttt aagcgatgtg tgcgtgcaaa 780gtattgcatt
ataattgcat gataactctt gtatgtttag agtcagtagg gcagggccgg
840ccggcagtca gacctggatt ggcgacacaa gtctgccagg acgactgcgg
tggcaaagtt 900ggtggagatt ttcgagttgg agctcctctg tgtctgtgtc
agacactcta actttcttgt 960ctcctgtttt ctgctttgcc tttcagcggc
cggttgcatt ggatgtacaa gtgtggcgtg 1020tggaaagcgc gcacgacacg
cgcgcgcgac gcccgccgcg gctggcacca gcctggcgct 1080ggatccaatc
tgctgcacgc cgcgcataaa tgcaagtgtc tactgtgata ttggcataat
1140ttaaacactc cccactggct cactaggact attgctgctc gcaagcccgt
cgcacagtta 1200acc 120321113DNAArtificialvector 2atgtcggtcg
gatttctggt cctcgcgctg ggcgcgcttg tcgtggcgac ggcggactac 60aaggacgacg
acgacaagca gcccacgacg actggcactc gcttcgaggg tttctcttac
120gcgggcaatg tcattggcta tgtgaacatg acgatggact actgcgacat
caaggccgcc 180atggctgctg gcaacttcac cgaggctctg tccatctact
ctactggcaa gaactcgttc 240tctggcctgg cgcgccgcac cttcttccgc
ttcgcctcgt acatcaccgc caacggctcc 300gtggagccgc tgcacgactc
catcctggcc ggcaaggaca cgtcctccct ggacgccgcc 360atccgggctg
ccctggccga cggcaaggcc accctggccg ccggtctgat ccaggtgggc
420acgctcaagt accacctgca cgaggtggat gaggcctaca acaagatcaa
gacctacctg 480gctgacggca ccggcaacct caccaacctg gtttctgacg
cctcgggcgc accccacaac 540gtggatgagg cctgggcgct gtgggccggc
ggcgccgcca acaactgagg caccctgtcc 600ggctgggcct cctccctggg
cgccgccatg ggcaccacct tcctcggcaa gagctatgtc 660aacaccgcca
tgatcaacac cgtcaatgag atgctggccg ccgcccgcct gtctaccctg
720aacatccaag cctacgacgc cgcgcgcacg aacgaggtcc gactgctgac
cctgctgggc 780ctgcagggcg tgtccgtggc cgcgtacacc gctgacgccg
ccgccgcctg caagcgcccc 840gccgccgagg tggaggatgc caagaccatg
atcgccgtgc actgggcgta cctggagcct 900atgctcaagc tgcgcaactt
caaggcctcc gccgtgaccg agctgcacca ccagctcacc 960gcctccaagc
tgagctacaa gaaggtggcc gccgcggtga agggcgtgct gtcggctatg
1020ggccgccgct ccagcgagct gggtgccccg cagtccgcca tcattgccgc
caactggaag 1080tgcagctcca agaccctgcg cagcattgcg taa
11133872DNAartificialvector 3agtgcgcctg ctgagcactg cttgcggagc
ctctttcgtg aaaagcgcgt ccgtatggtt 60agatagcagt gctctggagc aggtctggtg
catggttgga tgatcgggta tgtgcatatc 120atgcgggcaa cacactcgct
ttctaggttg ctgatggagc caaccttgct gaagtgtagt 180gtgtgcatgc
agagagtgca cagcaggata gccttctggc ggaatgctgc gctgcttcat
240cacatgtagt gcgtggtaac cgcgggatac ggcgtccagc cccgatttgt
caagtttggt 300cgtggcgttc tggcatacat tgtttgcact ctgtgaagcg
ctttcggccc caaaggttca 360tcacctaagg aactgcaagt gcatggatgc
cagaaggact gcgccaatgg ctttcgttta 420tgacggtcct ggctagaatg
cttggcttgg acccgagcta cagaaagctg cagcttgata 480ttggactggt
ggcaggtgca ggagggtaag ggcttagacc acaaggtgtg tgagtagcag
540atggagcgcg gactaatcta ggtgatttag ggcgcggtgg agggtgaggt
ggttggcgct 600tcggttctcg ggtctgactt tttgcaggtg gcatattcaa
tagcacttgc aggtatgaga 660gctagacggc ccgtatcagg catctgccgt
gcgggttgca atgtggagat gtacatgcag 720gcagcaggca gcaagtagcg
gagtcaaggc tgtgtgatac acgcagggta cgtgcaaggc 780acgggagcgt
gctgccagag ggacctagca gatcaacaat tggaaccaaa ctggcaggtt
840ttggtttgat tgtgcaagag tttgcgcgct gc 872424DNAArtificialVector
4gactacaagg acgacgacga caag 2458PRTArtificialepitope 5Asp Tyr Lys
Asp Asp Asp Asp Lys 1 5 62686DNAArtificialVector 6tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
420cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata
gctgtttcct 480gtgtgaaatt gttatccgct cacaattcca cacaacatac
gagccggaag cataaagtgt 540aaagcctggg gtgcctaatg agtgagctaa
ctcacattaa ttgcgttgcg ctcactgccc 600gctttccagt cgggaaacct
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660agaggcggtt
tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
720gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
gttatccaca 780gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
gccagcaaaa ggccaggaac 840cgtaaaaagg ccgcgttgct ggcgtttttc
cataggctcc gcccccctga cgagcatcac 900aaaaatcgac gctcaagtca
gaggtggcga aacccgacag gactataaag ataccaggcg 960tttccccctg
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
1020ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
ctgtaggtat 1080ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
tgcacgaacc ccccgttcag 1140cccgaccgct gcgccttatc cggtaactat
cgtcttgagt ccaacccggt aagacacgac 1200ttatcgccac tggcagcagc
cactggtaac aggattagca gagcgaggta tgtaggcggt 1260gctacagagt
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
1320atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
ttgatccggc 1380aaacaaacca ccgctggtag cggtggtttt tttgtttgca
agcagcagat tacgcgcaga 1440aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc tcagtggaac 1500gaaaactcac gttaagggat
tttggtcatg agattatcaa aaaggatctt cacctagatc 1560cttttaaatt
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
1620gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
atttcgttca 1680tccatagttg cctgactccc cgtcgtgtag ataactacga
tacgggaggg cttaccatct 1740ggccccagtg ctgcaatgat accgcgagac
ccacgctcac cggctccaga tttatcagca 1800ataaaccagc cagccggaag
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860atccagtcta
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
1920cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
tggtatggct 1980tcattcagct ccggttccca acgatcaagg cgagttacat
gatcccccat gttgtgcaaa 2040aaagcggtta gctccttcgg tcctccgatc
gttgtcagaa gtaagttggc cgcagtgtta 2100tcactcatgg ttatggcagc
actgcataat tctcttactg tcatgccatc cgtaagatgc 2160ttttctgtga
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
2220agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
aactttaaaa 2280gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
caaggatctt accgctgttg 2340agatccagtt cgatgtaacc cactcgtgca
cccaactgat cttcagcatc ttttactttc 2400accagcgttt ctgggtgagc
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460gcgacacgga
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
2520cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
taaacaaata 2580ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
tctaagaaac cattattatc 2640atgacattaa cctataaaaa taggcgtatc
acgaggccct ttcgtc 2686
* * * * *