U.S. patent number 7,892,786 [Application Number 10/566,886] was granted by the patent office on 2011-02-22 for methods for expression and purification of immunotoxins.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Yuan-Yi Liu, David M. Neville, Jung-Hee Woo.
United States Patent |
7,892,786 |
Neville , et al. |
February 22, 2011 |
Methods for expression and purification of immunotoxins
Abstract
The present invention relates to a method of expressing an
immunotoxin in Pichia pastoris strain mutated to toxin resistance
comprising a) growing the Pichia pastoris in a growth medium
comprising an enzymatic digest of protein and yeast extract and
maintaining a dissolved oxygen concentration at 40% and above; and
b) performing methanol induction with a limited methanol feed of
0.5-0.75 ml/min/IO L of initial volume during induction along with
a continuous infusion of yeast extract at a temperature below
17.5.degree. C., antifoaming agent supplied up to 0.07%, agitation
reduced to 400 RPM, and the induction phase extended out to 163
h.
Inventors: |
Neville; David M. (Bethesda,
MD), Woo; Jung-Hee (Rockville, MD), Liu; Yuan-Yi
(Potomac, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
34115570 |
Appl.
No.: |
10/566,886 |
Filed: |
August 2, 2004 |
PCT
Filed: |
August 02, 2004 |
PCT No.: |
PCT/US2004/024786 |
371(c)(1),(2),(4) Date: |
February 01, 2006 |
PCT
Pub. No.: |
WO2005/012495 |
PCT
Pub. Date: |
February 10, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060216782 A1 |
Sep 28, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60491923 |
Aug 1, 2003 |
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Current U.S.
Class: |
435/69.1;
536/24.1; 435/254.23; 536/23.7; 435/320.1; 435/69.7; 435/483 |
Current CPC
Class: |
C07K
1/18 (20130101); A61K 47/6829 (20170801); C12N
1/16 (20130101); C07K 16/2809 (20130101); A61K
47/6849 (20170801); C07K 2319/00 (20130101); C07K
2317/622 (20130101) |
Current International
Class: |
C12N
15/09 (20060101); C12N 15/10 (20060101); C12N
15/81 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
McGrew et al, Expression of trimeric CD40 ligand in Pichia
pastoris: use of a rapid method to detect high-level expressing
transformants, Gene, 1997, vol. 187 (2), pp. 193-200. cited by
examiner .
Jahic, M. et al., "Temperature limited fed-batch technique for
control of proteolysis in Pichia pastoris bioreactor cultures,"
Microbial Cell Factories, 2:11p (Jun. 2003). cited by other .
Jahic, Mehmedalija et al., "Analysis and control of proteolysis of
a fusion protein in Pichia pastoris fed-batch processes," Journal
of Biotechnology, 102(1):45-53, Apr. 2003). cited by other .
Li, Zhengjun et al. "Low-temperature increases the yield of
biologically active herring antifreeze protein in Pichia pastoris,"
Protein Expression and Purification, 21(3):438-445 (Apr. 2001).
cited by other .
Liu, Y. Y. et al., "Targeted introduction of a diphtheria toxin
resistant mutation into the chromosomal EF-2 locus of Pichia
pastoris and expression of immunotoxin in the EF-2 mutants,"
Protein Expression and Purification, 30(2):262-274 (Aug. 2003).
cited by other .
Sarramegna, V. et al., "Optimizing Functional versus Total
Expression of the Human mu-Opioid Receptor in Pichia pastoris,"
Protein Expression and Purification, 24(2):212, 2002. cited by
other .
Woo, Jung Hee et al., "Gene Optimization is necessary to express a
bivalent anti-human anti-T cell immunotoxin in Pichia pastoris,"
Protein Expression and Purification, 25(2):270-282 (Jul. 2002).
cited by other .
Woo, Jung Hee et al., "Increasing secretion of a bivalent
anti-T-cell immunotoxin by Pichia pastoris," Applied and
Environmental Microbiology, 70(6):3370-3376 (Jun. 2004). cited by
other.
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Primary Examiner: Marvich; Maria
Attorney, Agent or Firm: Corless; Peter F. McKiernan;
Colleen Edwards Angell Palmer & Dodge LLP
Government Interests
The U.S. Government has certain rights in this invention.
Parent Case Text
This application claims benefit of U.S. Provisional Application
60/491,923 filed Aug. 1, 2003.
Claims
What is claimed is:
1. A method of expressing an immunotoxin in a Pichia pastoris that
expresses the immunotoxin, the method comprising: a) growing the
Pichia pastoris in a growth medium comprising an enzymatic digest
of protein and yeast extract wherein the immunotoxin coding
sequence is under control of an AOX1 promoter; and b) performing
methanol induction on the cultured Pichia pastoris, wherein the
methanol induction is performed at a temperature comprising
17.5.degree. C. or below.
2. The method of claim 1, wherein the methanol induction comprises
a limited methanol feed comprising administration of methanol at a
concentration of between 0.5-0.75 ml/min (per 10 L initial growth
medium).
3. The method of claim 1, wherein the methanol induction comprises
administration of a methanol and glycerol containing feed.
4. The method of claim 3, wherein the ratio of methanol to glycerol
in the methanol and glycerol containing feed is about 4:1.
5. The method of claim 1, wherein the immunotoxin is a fusion
protein.
6. The method of claim 1, wherein the immunotoxin comprises a
diphtheria toxin moiety.
7. The method of claim 6, wherein the diphtheria toxin moiety is
truncated.
8. The method of claim 7, further comprising a CD3 antibody
moiety.
9. The method of claim 8, wherein the immunotoxin comprises
A-dmDT390-bisFv(G.sub.4S).
10. The method of claim 6, wherein the Pichia pastoris comprises a
mutation in the amino acid sequence of the diphthamide region of
EF-2, wherein the mutation prevents ADP ribosylation of EF-2.
11. The method of claim 10, wherein the mutation is a substitution
from Glycine to Arginine at position 701 of the amino acid sequence
encoded by SEQ ID NO: 13.
12. The method of claim 1, wherein the Pichia pastoris comprises a
mutation in the amino acid sequence of the diphthamide region of
EF-2, wherein the mutation prevents ADP ribosylation of EF-2.
13. The method of claim 1, wherein the enzymatic digest of protein
is an enzymatic digest of soy protein.
14. The method of claim 1, further comprising contacting the Pichia
pastoris with phenylmethanesulfonyl fluoride and a source of amino
acids.
15. The method of claim 14, wherein the Pichia pastoris is
contacted with the phenylmethanesulfonyl fluoride and the source of
amino acids for at least 2 hours during the methanol induction.
16. The method of claim 14, wherein the phenylmethanesulfonyl
fluoride is dissolved in a 4:1 methanol:glycerol induction feed and
the concentration of phenylmethanesulfonyl fluoride does not exceed
10 mM.
17. The method of claim 14, wherein the source of amino acids is a
yeast extract.
18. The method of claim 1, wherein the temperature can be selected
from the group of temperatures consisting of 17.5, 17.0, 16.5,
16.0, 15.5, 15.0, 14.5, 14.0, 13.5, 13.0, 12.5, and 12.0.degree.
C.
19. The method of claim 1, wherein the temperature is about
15.degree. C.
20. The method of claim 1, wherein the composition of the growth
medium is about 4% glycerol, about 2% yeast extract, about 2%
enzymatic digest of soy protein, about 1.34% yeast nitrogen base
with ammonium sulfate and without amino acids, and about 0.43% PTM1
solution.
21. The method of claim 20, wherein the growth medium further
comprises an antifoaming agent.
22. The method of claim 21, wherein the antifoaming agent is at a
concentration of about 0.01% or greater.
23. The method of claim 22, wherein the composition of the growth
medium is about 4% glycerol, about 2% yeast extract, about 2%
enzymatic digest of soy protein, about 1.34% yeast nitrogen base
with ammonium sulfate and without amino acids, about 0.43% PTM1
solution and about 0.02% antifoaming agent.
24. The method of claim 1, wherein dissolved oxygen concentration
in the growth medium is maintained at a value of 40% or higher.
25. The method of claim 1, wherein the growth step is at a pH of
about 3.5 and the methanol induction step is at a pH of about
7.0.
26. The method of claim 1, wherein the methanol induction step is
performed for between about 22 and 288 h.
27. The method of claim 1, wherein the induction is performed for
at least 4 hours and the temperature is ramped down from 28.degree.
C. during the growth phase to 15.degree. C. during the first four
hours of methanol induction.
28. The method of claim 1, wherein the induction step is carried
out at 17.5.degree. C. or below for at least 44 hours.
29. The method of claim 28, wherein the induction step is carried
out at 17.5.degree. C. or below for at least 67 hours.
30. A method of expressing an immunotoxin in a Pichia pastoris that
expresses the immunotoxin, the method comprising: a) growing the
Pichia pastoris in a growth medium comprising an enzymatic digest
of protein and yeast extract wherein the immunotoxin coding
sequence is under control of an AOX1 promoter; and b) performing
methanol induction on the Pichia pastoris, wherein the methanol
induction comprises a limited methanol feed comprising
administration of methanol at a concentration of 0.5-0.75 ml/min/10
L of initial volume of the growth medium, wherein the induction is
performed at a temperature comprising 17.5.degree. C. or below,
further comprising an antifoaming agent in the growth medium at a
concentration of up to 0.07%, wherein agitation is maintained at
about 400 RPM during the induction step, and wherein the induction
step is performed for between about 22 and 288 h.
31. The method of claim 30, wherein the induction is performed for
at least 4 hours and the temperature is ramped down from 28.degree.
C. during the growth phase to 15.degree. C. during the first four
hours of methanol induction.
32. The method of claim 30, wherein the induction step is carried
out at 16.5.degree. C. or below for at least 44 hours.
33. The method of claim 32, wherein the induction step is carried
out at 16.5.degree. C. or below for at least 67 hours.
34. A method of expressing an immunotoxin in a Pichia pastoris that
expresses the immunotoxin, the method comprising: a) growing the
Pichia pastoris in a growth medium comprising about 4% glycerol,
about 2% yeast extract, about 2% enzymatic digest of soy protein,
about 1.34% yeast nitrogen base with ammonium sulfate and without
amino acids, and about 0.43% PTM1 solution wherein the immunotoxin
coding sequence is under control of an AOX1 promoter, wherein the
growth occurs at a pH of about 3.5, and wherein the dissolved
oxygen concentration in the growth medium is maintained at a value
of 40% or higher; and b) performing methanol induction on the
Pichia pastoris, wherein the methanol induction comprises a limited
methanol feed comprising administration of methanol at a
concentration of 0.5-0.75 ml/min/10 L of initial volume of growth
medium, wherein the induction is performed at a temperature of
15.degree. C., wherein the pH of the growth medium during the
induction step is about 7.0, further comprising an antifoaming
agent at a concentration of 0.02%, wherein the agitation is
maintained at about 400 RPM during the induction step, and wherein
the induction step is performed for about 163 h.
35. The method of claim 34, wherein the induction temperature is
ramped down to during the first four hours of methanol
induction.
36. The method of claim 34, wherein the induction step is carried
out at 15.degree. C. or below for at least 44 hours.
37. The method of claim 36, wherein the induction step is carried
out at 15.degree. C. or below for at least 67 hours.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods of protein
expression and purification, and more specifically, to methods of
expression and purification of immunotoxins.
2. Description of the Related Art
The number of organ transplants performed in the United States each
year is approximately 24,000 and consists predominantly of kidney
transplants (14,000), liver transplants (5,000), heart transplants
(2,200), and smaller numbers of pancreas, lung, heart-lung, and
intestinal transplants (2002 OPTN/SRTR Annual Report).
Transplant tolerance remains an elusive goal for patients and
physicians whose ideal would be to see a successful, allogenic
organ transplant performed without the need for indefinite,
non-specific maintenance immunosuppressive drugs and their
attendant side effects. Many of these patients have been treated
with cyclosporin, azathioprine, and prednisone with a variety of
other immunosuppressive agents being used for induction or
maintenance immunosuppression. The average annual cost of
maintenance immunosuppressive therapy in the United States is
approximately $11,000 (Immunosuppressive Drugs Coverage Act,
National Kidney Foundation, available at
www.kidney.org/general/pubpol/immufact.cfm). While these agents are
effective in preventing rejection, the side effects of
immunosuppressive therapy are considerable. Immunosuppressive
therapy induces nonspecific unresponsiveness of the immune system.
Recipients are susceptible to infection and there is a risk of
malignancy such as in the form of post transplant
lymphoproliferative disorders. A major goal in transplant
imununobiology is the development of specific immunologic tolerance
to organ transplants with the potential of freeing patients from
the side effects of continuous pharmacologic immunosuppression and
its attendant complications and costs.
A bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G.sub.4S) was
developed for tolerance induction for transplantation, T-cell
leukemia and autoimmune diseases. The immunotoxin consists of the
first 390 amino acid residues of diphtheria toxin (DT390) and two
tandem antigen-binding domains (sFv) from the anti-CD3 antibody
UCHT1, that are responsible for binding the immunotoxin to the
CD3.epsilon..gamma. subunit of the T cell receptor complex. The
anti-CD3.epsilon. antibody moiety enables the immunotoxin to target
specific cells and the diphtheria toxin moiety kills the target
cells. The immunotoxin may be utilized to effect at least partial
T-cell depletion in order to treat or prevent T-cell mediated
diseases or conditions of the immune system.
Administration of an anti-T cell immunotoxin provides an approach
for specific immunologic tolerance. It is applicable to new organ
transplants and potentially to existing transplants in recipients
with stable transplant function. The immunotoxin can provide highly
specific immunosuppression and imparts transplant tolerance in
primates, without the adverse effects of nonspecific
immunosuppressive drugs, anti-lymphocyte serum or radiation. It is
a goal in this field to inhibit the rejection response to the point
that rejection is not a factor in reducing average life span among
transplant recipients.
The methylotrophic yeast Pichia pastoris has been used successfully
to express heterologous proteins from different origins (Gellissen
2000). As an eukaryote, Pichia pastoris has the ability to perform
many post-translational protein modifications such as proteolytic
processing, folding, disulfide bond formation and glycosylation.
Like other yeasts, Pichia pastoris offers significant advantages
over higher eukaryotic cells such as Chinese hamster ovary (CHO) or
baculovirus-infected insect cell expression systems. It is easy to
manipulate, has a rapid growth rate and requires inexpensive media.
These greatly reduce the production time and cost, especially on a
commercial scale. Unlike Saccharomyces cerevisiae, Pichia pastoris
is not a strong fermentor and can be easily cultured to very high
cell density of >100 g dry cell weight/liter (Siegel et al.,
1989). This, plus the strong AOX1 promoter employed in driving
transcription of foreign genes, have made Pichia pastoris the
system of choice for high levels of expression of heterologous
proteins. The AOX1 promoter also has advantages in the expression
of foreign proteins that are deleterious to the expressing host
because the promoter is tightly regulated and highly repressed
under non-methanolic growth conditions. The inducible and tightly
regulated AOX1 promoter has allowed successful expression of DT
based immunotoxins, in secreted form, in Pichia pastoris strains
without any mutation to confer a resistance to DT. (Woo et al.,
2002). However, diphtheria toxin (DT) is a very potent toxin to all
eukaryotic cells if its catalytic domain can find a route to the
cytosol. Pichia pastoris is inherently sensitive to these
toxins.
The prior art teaches methods for growing Pichia pastoris. For
example, Pichia pastoris may be grown in a fermentor. One protocol
for Pichia pastoris fermentation contains glycerol as the initial
carbon source, followed by brief carbon starvation and use of
methanol as the carbon source (Pichia pastoris Fermentation Using a
BioFlo 110 Benchtop Fermentor, New Brunswick Scientific).
Woo et al. disclosed that, when expressing a bivalent anti-human
anti-T cell immunotoxin A-dmDT390-bisFv(G.sub.4S) in Pichia
pastoris, a buffered complex medium at pH 7.0 with 1% casamino
acids provided the highest expression in shake flask culture and
that the expression level was improved by adding PMSF in the range
of 1 to 3 mM. (25 Protein Expression and Purification 270-82
(2002)).
Sreekrishma disclosed that an increased secretion level was
obtained using Pichia pastoris in shake flask cultures when the
cells were highly aerated and in a buffered medium at pH 6.0 that
was supplemented with yeast extract and peptone (Chapter 16,
Industrial Microorganisms: Basic and Applied Molecular Genetics
(1993)). The growth medium contained yeast nitrogen base with
ammonium sulfate, biotin and glycerol buffered to pH 6.0 with
potassium phosphate buffer as well as yeast extract and peptone.
The induction medium contained methanol in place of glycerol.
In contrast, the present invention provides an improved method of
using Pichia pastoris to produce an immunotoxin. The immunotoxins
expressed and purified in the present invention can be used in a
method of inducing immune tolerance. It would be desirable to
provide a method of expression and purification that increased the
yield of immunotoxins. The present invention addresses this problem
and others in the manner described below.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a method of
expressing an immunotoxin in Pichia pastoris toxin resistant EF-2
mutant comprising a) growing the Pichia pastoris in a growth medium
comprising an enzymatic digest of protein and yeast extract; and b)
performing methanol induction of the Pichia pastoris with a limited
methanol feeding of 0.5 to 0.75 ml/min (per 10 L initial medium)
during induction, and wherein the methanol induction is at a
temperature of below about 17.5.degree. C.
In another aspect, the present invention relates to a method of
expressing an immunotoxin in Pichia pastoris comprising a) growing
the Pichia pastoris in a growth medium comprising an enzymatic
digest of protein and yeast extract; and b) performing methanol
induction of the Pichia pastoris with a methanol and glycerol
containing feed, wherein the Pichia pastoris is contacted with a
phenylmethanesulfonyl fluoride and a source of amino acids and
wherein the methanol induction is at a temperature of below about
17.5.degree. C.
In yet another aspect, the present invention relates to a method of
purifying a non-glycosylated immunotoxin comprising a) loading a
solution containing the non-glycosylated immunotoxin onto a
hydrophobic interaction column; b) obtaining a first
non-glycosylated immunotoxin containing eluant from the hydrophobic
interaction column; c) loading the non-glycosylated immunotoxin
containing eluant from step (b) onto an anion exchange column; d)
obtaining a second non-glycosylated immunotoxin containing eluant
from the anion exchange column by eluting the non-glycosylated
immunotoxin with a sodium borate solution; e) diluting the
concentration of sodium borate in the second non-glycosylated
immunotoxin containing eluant from step (d) to about 50 mM or less;
f) concentrating the diluted non-glycosylated immunotoxin
containing eluant from step (e) over an anion exchange column; and
g) obtaining a purified non-glycosylated immunotoxin from the anion
exchange column.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention, as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings illustrate one or more embodiments of the
invention and, together with the written description, serve to
explain the principles of the invention.
FIG. 1 shows conservation of diphthamide domain and DT-resistant
mutations in eukaryotic EF-2s and nucleotide sequence mutations for
the substitution of Arg for Gly 701 in Pichia pastoris EF-2. The
underlined sequences are the site for the restriction enzyme Sac II
that resulted from the nucleotide mutations. See SEQ ID NOS:
1-10.
FIG. 2. The 5' end sequence of Pichia pastoris EF-2 showing the
short intron (SEQ ID NO: 11; mRNA) and (SEQ ID NO: 12; gDNA). The
5' splice site, branch site and 3' splice site are under lined.
EF-2 coding sequence is in bold.
FIG. 3. (A) (B) (C) (D) Nucleotide and deduced amino acid sequence
of Pichia pastoris EF-2 (SEQ ID NO: 13). The nucleotide sequence is
numbered from the beginning of the initiation codon. Consensus
GTP-binding motif in the protein sequence is AHVDHGKST (SEQ ID NO:
14), the threonine residue putatively phosphorylated in vivo by
EF-2 kinase is circled and the effector domain conserved among all
elongation factors is DEQERGITIKSTA (SEQ ID NO:15). The 22
well-conserved residues of the diphthamide domain are boxed.
FIG. 4. Targeted mutation using the 3' sequence of EF-2 that has
been mutated in vitro. The mutating plasmid pBLURA-.DELTA.5'mutEF-2
contains four essential elements: .beta.-lactamase gene (Ampr),
Uracil selection marker (URA3), 3'AOX1 transcription termination
sequence (TT) and the in vitro mutated FF-2 3' sequence,
.DELTA.5'mutEF-2.
FIG. 5. Agarose gel electrophoresis of PCR products of selected
Ura+ clone derived from Pichia pastoris JC308 strain. (a) PCR
products with primers 1 and M; (b) PCR products with primers 2 and
w; (c). Sca II digested PCR products with primers 2 and 3.
FIG. 6. Western blot analysis of cytosolic expression of DT-A chain
in mutated and wild type Pichia pastoris strains. (a) Lanes 1-6 are
the cell extracts of 6 independent clones of mutEF2JC307-8
transformed with pPIC3-DtA, +C: The purified A-dmDT390-bisFv. M:
SeeBlue plus2 Protein markers (Invitrogen). (b) Cytosolic
expression of DT-A chain in cultures of two separated colonies of
mut-3 and mut-5 that are mutEF2JC307-8(3) and (5) respectively, C3
and C4. Protein samples are loaded on 4-12% NuPAGE gels
(Invitrogen).
FIG. 7. The effect of intra-cellular expression of DT-A on the
survival of Pichia pastoris strains with mutated or wild type EF-2.
Mut-3 and Mut 5 are EF-2 mutants mutEF2JC307-8-DtA(3) and (5)
respectively, Mut-3 expressed DT A chain in the cytosol, mut-5 did
not. C3 and C4 are the wild type EF-2 strains that did (C4) or did
not express DT A chain in the cytosol. The first bar in each
category indicates the colony-forming units before methanol
induction. The second bar in each category represents the
colony-forming units after methanol induction.
FIG. 8. Schematic presentation of plasmid construction. (a).
pBLARG-A-dmDT390-bisFv; (b). pPGAPArg-A-dmDT390-bisFv; (c).
pPGAPHis-A-dmDT390-bisFv.
FIG. 9. Western blot analysis of expression of A-dmDT390-bisFv.
Samples of culture media (a) and cell extracts (b) were loaded on
4-12% NuPAGE gels (Invitrogen). Lanes of +c are purified
A-dmDT390-bisFv. Lanes 1-9 were samples of 9 selected clones of mut
EF2JC303 transformed with 2 copies of the A-dmDT390-bisFv gene.
Lanes 10, 11 and 12 were samples of single copy clones: lane 12 was
the non-mutated EF-2 clone JHW#2, lanes 10 and 11 were two of
selected clones of mutEF2JC307-8(1) and mutEF2JC307-8(2) that is
also called YYL#8-2.
FIG. 10. Comparison of the methanol consumption rate among
different Pichia pastoris strains. All of these strains are Mut+
(Methanol utilization plus) except for pJHW#3, which is MutS
(Methanol utilization slow). pJHW#2 to 5 and the EF-2 mutant
YYL#8-2 all expressed the bivalent immunotoxin A-dmDT390-bisFv.
X-33 is a wild type strain that does not express A-dmDT390-bisFv,
but was transformed with the expression vector.
FIG. 11. Comparison of profiles of methanol consumption rate
between X-33 and JW102 and between different nutrient feeding of
JW102 at the indicated temperature. YE and casa represents feeding
of yeast extract and casamino acids, respectively.
FIG. 12 Lowering agitation speed in fermentation reduces
immunotoxin aggregates. Fermentation performed at high agitation
speed resulted in more than 50% of the secreted immunotoxin being
present in inactive aggregate forms in the supernatants. In
addition, aggregates accumulated over induction time. However,
lowering agitation speed from 800 rpm to 400 rpm reduced
immunotoxin aggregates. Immunotoxin aggregates were maintained at
the same level over induction time.
FIG. 13 Effect of TWEEN 20.RTM. on aggregation of purified
immunotoxin after 20 hrs incubation at 30.degree. C. at 250 rpm.
Using purified immunotoxin, TWEEN 20.RTM. prevented the formation
of aggregates by agitation. Approximately 50% of the purified
immunotoxin was aggregated by incubation at 30.degree. C. at 250
rpm for 20 hours. However, 0.01%-0.04% of TWEEN 20.RTM.
significantly reduced the aggregation purified immunotoxin by
agitation.
FIG. 14. Change of gain of wet cell density during the first 44
hours of methanol induction. MeOH, methanol alone and feeding of
casamino acids; M: G=4:1, methanol/glycerol mixed feeding and
feeding of casamino acids; YE+MeOH, feeding of yeast extract and
methanol alone; YE+4:1, feeding of yeast extract and
methanol/glycerol mixed feeding.
FIG. 15. Expression level of the bivalent immunotoxin and its final
purification yield depending on induction temperature. A: change of
expression level by induction temperature. B: change of the final
purification yield from 1 liter of supernatant taken at 22, 44, and
67 hours of methanol induction. 22 hrs, 44 hrs, and 67 hrs
represent time of methanol induction. C: change of methanol
consumption depending on induction temperature.
FIG. 16. A representative of optimized fermentation runs. Samples
taken at indicated induction time points were fractionated on 4-20%
SDS-tris-glycine gel and the gel was stained with Coomassie blue
dye. Arrow indicates the position of the bivalent immunotoxin. Mark
12 marker (Invitrogen) was used.
FIG. 17. SDS-PAGE analysis of proteins obtained by butyl 650M
capture step. Lane 1.about.4, sample flow-through fraction
#1.about.#4; lane 5, pooled sample flow-through fractions; lane
6.about.8, wash fraction #1.about.#3; lane 9, pooled wash
fractions; lane 10, 11, 17, supernatant; lane 12, Mark 12 protein
standards (Invitrogen); lane 13.about.15, eluted fraction #1##3;
lane 16, pooled eluted fractions. IT, immunotoxin.
FIG. 18. SDS-PAGE analysis of proteins obtained by Poros 50 HQ
borate anion exchange step. Lane 1, Mark 12 protein standards
(Invitrogen); lane 2, sample obtained from Butyl 650M HIC step;
lane 3.about.7, sample flow-through fraction #1.about.#5; lane 8,
fraction #1 eluted with 25 mM borate in Buffer B; lane 9, fraction
#2 eluted with 50 mM borate in Buffer B; lane 10, fraction #3
eluted with 75 mM borate in Buffer B; lane 11, fraction #4 eluted
with 100 mM borate in Buffer B; lane 12, fraction #5 fraction
eluted with 1 M NaCl in Buffer B. IT, immunotoxin.
FIG. 19. Analytical gel filtration and SDS-PAGE analysis of
purified immunotoxin. A: Chromatogram of Superdex 200 10/300 GL gel
filtration. B: Picture of Coomassie-stained SDS-polyacrylamide
gel.
FIG. 20. (a) (b) (c) Amino acid sequence of Ala-dmDT390bisFv(UCHT1)
(SEQ ID NO:16).
FIG. 21. Comparison of profiles of cell growth, methanol
consumption and immunotoxin secretion during methanol induction.
Panel A. X-33 strain and the immunotoxin producing toxin resistant
EF-2 mutant mutEF2JC307-8(2). These two strains had similar profile
of methanol consumption rate and wet cell density gain during
methanol induction. The data shown in panel A was for X-33. For the
toxin resistant mutant, the maximum methanol consumption rate and
wet cell density gain at 44 h of methanol induction was 2.2 ml/min
and 9.17%, respectively. Panels B-F. strain JW102. Constant
conditions in all panels A-F were a glycerol batch phase followed
by a glycerol-fed batch phase prior to induction. For induction,
either pure methanol (MeOH) alone or 4:1 methanol:glycerol (M/G)
mixed feed was used. PMSF at 10 mM in methanol was infused
continuously during induction. Casamino acid feeding was performed
when yeast extract (YE) feeding was not done. Induction conditions:
panel A, M/G feeding and no YE feeding; panel B, methanol feeding
and no YE feeding; panel C, methanol feeding and YE feeding; panel
D, M/G feeding and YE feeding; panel E, M/G feeding and no YE
feeding; panel F, M/G feeding and YE feeding. The induction
temperature was 23-25.degree. C. in A-E and 15.degree. C. in F
(note the right hand axis in panel F is compressed 2-fold compared
to the other panels). Methanol consumption rate (ml/min), dotted
line; wet cell density (%, w/v), solid line; and level of secreted
immunotoxin (mg/L), dashed line. Because of the large amount of
work involved in 10 L bioreactor fermentations, it was not
practical to replicate the results in panels A-E. The optimized
method, panel F, was performed 3 times and the points are averages
with standard error of the mean shown when greater than 10%. The
actual data points for wet cell density and level of secreted
immunotoxin are shown as squares and circles. The actual data
points for methanol consumption rate are omitted because methanol
consumption rate was measured every minute.
FIG. 22. Protein degradation and immunotoxin production. A. Time
course of immunotoxin levels during methanol induction in cultures
with methanol and yeast extract feeding (FIG. 21C). The immunotoxin
band is marked with IT and an arrow. B. Analysis of residual
immunotoxin by SDS-PAGE after incubation (28.degree. C., 20 h, and
250 rpm shaking) of an equal volume of purified immunotoxin (250
.mu.g/ml) with an equal volume of the supernatants collected at the
indicated times following methanol induction. Mixtures of equal
volumes of purified immunotoxin and PBS buffer or supernatant from
0 h, were used as the controls (CON). Ten .mu.l of the prepared
samples were loaded for SDS-PAGE and fractionated on 4-20%
SDS-tri-glycine gels under non-reducing conditions. Gels were
stained with Coomassie blue dye. Mark12 marker (Invitrogen) was
used as the protein marker.
FIG. 23. Effect of temperature on immunotoxin production. Samples
taken at indicated induction time points (44, 50, 67 h) from runs
at different induction temperature (15.about.23.degree. C.) were
fractionated on 4-20% SDS-tris-glycine gels under non-reducing
condition. Continuous feeding of yeast extract and
methanol-glycerol feed were used for all runs. The gels were
stained with Coomassie blue dye. IT-dp-degraded products of the
bivalent immunotoxin. These degradation products were identified by
Western blots using anti-DT antibody and anti-(G.sub.4S).sub.3
linker antibody. The anti-(G.sub.4S).sub.3 linker antibody could
detect the bivalent immunotoxin and degraded products, because the
immunotoxin contained three (G.sub.4S).sub.3 linkers. Arrows point
to bands not related to the bivalent immunotoxin. IT--bivalent
immunotoxin. Mark12 marker (Invitrogen) was used as the protein
marker.
FIG. 24. Analysis of protease activity in supernatants in the
absence and presence of PMSF during methanol induction at
15.degree. C. Supernatants were taken at 0, 22, 44 and 67 h of
methanol induction from fermentation runs treated with continuous
feeding of yeast extract and methanol-glycerol feed. The
supernatants were incubated with unnicked CRM9 as the substrate.
After incubation, 10 .mu.l of the sample was fractionated on 4-20%
SDS-tris-glycine gels under reducing conditions. After staining and
drying, the gel was digitized and analyzed for band intensity of
unnicked CRM9 by using NIH Image software. PMSF supplementation
during methanol induction, solid line; no PMSF, dotted line. Each
data point is the average from 3 fermentation runs with the
standard error of the mean.
DETAILED DESCRIPTION OF THE INVENTION
Before the present compounds, compositions, articles, devices,
and/or methods are disclosed and described, it is to be understood
that they are not limited to specific synthetic methods or specific
recombinant biotechnology methods unless otherwise specified, or to
particular reagents unless otherwise specified, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting.
As used in the specification and the appended claims, the singular
forms "a," "an" and "the" include the plural forms unless the
context clearly dictates otherwise. Thus, for example, reference to
"a pharmaceutical carrier" includes mixtures of two or more such
carriers, and the like.
Ranges can be expressed herein as from "about" one particular
value, and/or to "about" another particular value. When such a
range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15.
In this specification and in the claims which follow, reference
will be made to a number of terms which shall be defined to have
the following meanings:
"Optional" or "optionally" means that the subsequently described
event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
"Contacted" means one substance is placed in physical association
with another substance.
"Non-glycosylated" means in the absence of glycosylation or in the
absence of glycosylation perceptible using routine methods known in
the art for measuring glycosylation. Thus non-glycosylated includes
having had glycosylation sites mutated so that glycosylation does
not occur, expressing in a system in which glycosylation will not
occur or not possessing glyscosylation sites in the wild type
state.
"Loading" a column means placing the sample in a position in which
at least a portion of the sample will eventually enter the part of
the column occupied by the resin.
An "enzymatic digest" refers to hydrolysis of a protein or peptide
at peptide bonds by one or more of various enzymes. Such enzymes
may include but are not limited to trypsin, chymotrypsin, pepsin,
thrombin, papain, bromelain, thermolysin, subtilisin or
carboxypeptidase A.
"Yeast extract" is a preparation of peptides and amino acids
obtained by proteolysis of the proteins within yeast cells.
"Induction" refers to providing a signal to a given promoter to
cause expression of a given gene.
"Feed" refers to providing fresh media or nutrients at a rate that
at least partially replaces the media or nutrients as they are
depleted.
"Moiety" refers to one portion of a molecule or compound that is
divided into multiple portions. In the present invention, moiety
may refer a toxin portion or an antibody portion of an
immunotoxin.
Throughout this application, the term "bivalent" is used to refer
to the ability of a single composition to bind two ligands. For
example, an A-dmDT390-bisFv(G.sub.4S) immunotoxin can bind two CD3
molecules. It is also understood that the term "divalent" can have
a similar meaning in the art. Herein, the terms "bivalent" and
"divalent" refer to the same property and are used
interchangeably.
The invention provides a system for expressing and purifying mutant
ADP ribosylating toxins and toxin fusion proteins in a Pichia
pastoris mutant. The methods of the present invention possess the
advantage of being compliant with Good Manufacturing Practices.
As used throughout, optionally, the immunotoxin is a fusion
protein. The immunotoxin can comprise a diphtheria toxin moiety. It
is understood and herein contemplated that other ADP ribosylating
immunotoxins may be used in the present methods. For example,
specifically contemplated are fusion proteins wherein the
immunotoxin comprises a Psuedomonas exotoxin A moiety. The toxin
moiety can be a truncated moiety and/or can comprise mutations as
compared to the wild-type toxin. The immunotoxin can further
comprise a CD3 antibody moiety or other antibody moiety. It is also
understood that the immunotoxin can comprise a targeting antibody
moiety other than the CD3 antibody. One of skill in the art would
know which moiety to use with the immunotoxin based on the target
cell. For example, a CD22 antibody may be used to direct the
immunotoxin fusion protein to B cells.
The invention provides a method of expressing an immunotoxin in
Pichia pastoris toxin resistant EF-2 mutant comprising growing the
Pichia pastoris in a growth medium comprising an enzymatic digest
of protein and yeast extract; and performing methanol induction at
a temperature of below about 17.5.degree. C.
In one aspect, the invention provides a method of expressing an
immunotoxin in Pichia pastoris toxin resistant EF-2 mutant
comprising growing the Pichia pastoris in a growth medium
comprising an enzymatic digest of protein and yeast extract; and
performing methanol induction of the Pichia pastoris with a limited
methanol feeding during induction of 0.5 to 0.75 ml/min (per 10 L
initial medium), wherein the methanol induction is at a temperature
of below about 17.5.degree. C.
Alternatively, the invention provides a method of expressing an
immunotoxin in Pichia pastoris comprising growing the Pichia
pastoris in a growth medium comprising an enzymatic digest of
protein (e.g., soy protein) and yeast extract; and performing
methanol induction of the Pichia pastoris with a methanol and
glycerol containing feed (e.g., with a methanol to glycerol ration
of about 4:1), wherein the Pichia pastoris is contacted with a
phenylmethanesulfonyl fluoride and a source of amino acids (e.g., a
yeast extract) and wherein the methanol induction is at a
temperature of below about 17.5.degree. C.
The act of contacting Pichia pastoris with phenylmethanesulfonyl
fluoride and the source of amino acids in the expression method
includes contacting the cells with phenylmethanesulfonyl fluoride
and the source of amino acids for at least 2 hours, including 2, 3,
4, 5, 6, 7, 8, 9, 10, or more hours or any amount in between.
Preferably, the phenylmethanesulfonyl fluoride is dissolved in the
4:1 methanol glycerol induction feed and the concentration does not
exceed 10 mM.
The methanol induction temperature is preferably below about 17.5,
and even more preferably is about 15.degree. C. Other temperatures
at which methane induction can take place in the practice of the
present method include 17.0, 16.5, 16.0, 15.5, 14.5, 14.0, 13.5,
13, 12.5, 12.degree. C. or any amounts in between.
Growth medium refers to any substance required for growth of the
selected organism. Substances required for growth may include but
are not limited to carbon, hydrogen, oxygen, nitrogen, phosphorus,
sulphur, potassium, magnesium, calcium, sodium, iron, trace
elements and organic growth factors. Various materials may be
included in growth medium to provide the required substances. Such
substances include but are not limited to simple sugars, extracts
such as peptone, soytone, tryptone, yeast extract, carbon dioxide,
vitamins, amino acids, purines and pyrimidines. An example of the
method of the invention utilizes the presence of an enzymatice
digest of soy produced by DIFCO. Another example of the method of
the invention utilizes the presence of yeast extract produced by
DIFCO. It is understood that yeast extracts and enzymatic digests
produced by any manufacturer of such items, for example, New
England Biosciences can be used.
In a specific non-limiting example of the method, the composition
of the growth medium is about 4% glycerol, about 2% yeast extract,
about 2% enzymatic digest of soy protein, about 1.34% yeast
nitrogen base with ammonium sulfate and without amino acids, and
about 0.43% PTM1 solution. Optionally, the growth medium further
comprises an antifoaming agent. More specifically, the antifoaming
agent is present at a concentration of about 0.01% or greater. For
example, the anti foaming agent can be present at a concentration
of 0.07% or any amount between about 0.01% and about 0.07%. The
optimum level of antifoaming reagent is chosen as the minimum
amount required to reduce the layer of foam above the liquid-air
interface to 1/2 inch or less. Thus, the composition of the growth
medium can be about 4% glycerol, about 2% yeast extract, about 2%
enzymatic digest of soy protein, about 1.34% yeast nitrogen base
with ammonium sulfate and without amino acids, about 0.43% PTM1
solution and about 0.02% antifoaming agent.
It is understood that one of skill in the art will know that the
composition of the growth medium may be altered to optimize for
maximal growth. Specifically contemplated are changes up to 20%
above or below the percentages of the components in the growth
medium. Thus herein disclosed is a growth medium, wherein the
composition of the growth medium is about 3.2%-4.8% glycerol, about
1.6%-2.4% yeast extract, about 1.6-2.4% enzymatic digest of soy
protein, about 1.07-1.61% yeast nitrogen base with ammonium sulfate
and without amino acids, and about 0.34%-0.52% PTM1 solution. For
example, specifically disclosed is a growth medium, wherein the
composition of the growth medium is about 3.6% glycerol, about 2.4%
yeast extract, about 1.9% enzymatic digest of soy protein, about
1.43% yeast nitrogen base with ammonium sulfate and without amino
acids, and about 0.43% PTM1 solution.
Optionally, the dissolved oxygen concentration in the growth medium
is maintained at a value of 40% or higher (e.g., 45%, 50%, 55%,
60%, or 65%) in the expression method of the invention. For
example, in the present invention, a glycerol-fed batch phase is
employed to obtain high cell density before initiation of methanol
induction. The glycerol-fed batch phase is started when the
dissolved oxygen rises above 40%. Glycerol is fed whenever the
dissolved oxygen rises above 40% and until the level drops below
40%. When the dissolved oxygen rises again after stopping glycerol
feeding, the feed is switched to methanol. A rise or spike in
dissolved oxygen, DO, level indicates exhaustion of the carbon
source. Typically the DO spike is used to indicate depletion of the
glycerol used for growth and indicates that a switch to methanol
for the induction phase should occur.
Furthermore, the growth step is optionally at a pH of about 3.0-4.0
and the methanol induction step is at a pH of about 6.7-7.4. For
example, the growth step is at a pH of 3.5 and the methanol
induction step is at a pH of 7.0.
Methanol induction time can be increased to maximize yields.
Typical induction times include 22 h, 44 h, 67 h, and 163 h.
Induction can be as long as 12 days (288 h). Thus, specifically
contemplated are methanol inductions that last about 22 h to about
12 days (288 h). For example, it is understood that methanol
induction can last 163 h.
Thus an embodiment of the present invention is a method of
expressing an immunotoxin in Pichia pastoris comprising a) growing
the Pichia pastoris in a growth medium comprising an enzymatic
digest of protein and yeast extract; b) performing methanol
induction of the Pichia pastoris, wherein the methanol induction
comprises a limited methanol feed of 0.5-0.75 ml/min/10 L of
initial volume, wherein the induction is performed at a temperature
below 17.5.degree. C., antifoaming agent supplied up to 0.07%, and
agitation is reduced to 400 RPM, and wherein the induction step is
performed for between about 22 and 288 h.
More specifically, an embodiment of the present invention comprises
a method of expressing an immunotoxin in Pichia pastoris comprising
a) growing a Pichia pastoris that expresses an immunotoxin in a
growth medium comprising about 4% glycerol, about 2% yeast extract,
about 2% enzymatic digest of soy protein, about 1.34% yeast
nitrogen base with ammonium sulfate and without amino acids, and
about 0.43% PTM1 solution, wherein the growth occurs at a pH of
about 3.5, and wherein the dissolved oxygen concentration in the
growth medium is maintained at a value of 40% or higher; and b)
performing methanol induction of the Pichia pastoris, wherein the
methanol induction comprises a limited methanol feed of 0.5-0.75
ml/min/10 L of initial volume, wherein the induction is performed
at a temperature is 15.degree. C., wherein the pH is about 7.0,
wherein antifoaming agent supplied at 0.02%, wherein the agitation
reduced to 400 RPM, and wherein the induction phase is about 163
h.
The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G.sub.4S),
which selectively kills human T cells, was developed for treatment
of T-cell leukemia, autoimmune diseases and tolerance induction for
transplantation (U.S. patent application Ser. No. 09/573,797,
incorporated by reference). The bivalent anti-T cell immunotoxin,
A-dmDT390-bisFv(G.sub.4S), consists of the first 390 amino acid
residues (DT390) of diphtheria toxin (DT) and two tandem
antigen-binding domains (sFv) from the anti-CD3 antibody UCHT1. Two
N-glycosylation sites in the DT390 immunotoxin have been removed by
introduction of two mutations (Liu et al., 2000), resulting in a
non-glycoprotein with a molecular weight of 96.5 kDa. The
immunotoxin can also comprise a linker molecule to join the
antibody moiety to the toxin moiety. The linker (L) can be a
Gly-Ser linker. The Gly-Ser linker can be but is not limited to
(Gly4Ser)n or (Gly3Ser)n. More specifically, the linker can be a
(Gly4Ser)3 linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 17), also referred
to herein as (G4S), or a (Gly3Ser)4 linker (GGGSGGGSGGGSGGGS) (SEQ
ID NO: 18), also referred to herein as (G3S). In a preferred
embodiment the immunotoxin comprises A-dmDT390-bisFv(G4S).
The immunotoxin is sensitive to pH levels below 6.0, as shown by
the fact that low pH induces an irreversible conformational change
in the translocation domain of the DT390 moiety. The translocation
domain mediates translocation of the A chain in the DT390 from the
endosomes or the plasma membrane to the cytosol in a proton
dependent manner. The catalytic A chain is responsible for protein
synthesis inhibition by ADP-ribosylation of elongation factor 2
(EF-2) in the cytosol. This inhibition of protein synthesis is
toxic to many eukaryotic cells. The pH sensitivity of the
immunotoxin restricts the use of cation exchange chromatography and
affinity chromatography based on eluting with a low pH buffer.
The use of toxin-resistant eukaryotic cells can overcome the
immunotoxin toxicity. However, selection and characterization of
toxin-resistant eukaryotic cells are tedious, labor intensive and
time-consuming work. Furthermore, the bivalent immunotoxin
production in a EF-2 mutant CHO cell expression system was limited
to 5 mg/L and could not be increased by selection for multiple gene
insertions. Due to this limitation, with three exceptions (12, 20,
25) all recombinant immunotoxin production for therapeutic uses has
been limited to E. coli production necessitating denaturation and
refolding from inclusion bodies (6). However, refolding of the
multi-domain structure of the bivalent immunotoxin from E. coli was
inefficient and full bioactivity was not recovered (25). Also, the
multi-domain structure of the bivalent immunotoxin hinders
efficient production in Escherichia coli. Therefore, the attempt to
develop a robust Pichia pastoris production system for the bivalent
immunotoxin was driven by the inadequacy of the existing
productions systems.
Pichia pastoris is a good expression system for the bivalent anti-T
cell immunotoxin A-dmDT390-bisFv as it provides optimal protein
folding compared to prokaryotic expression systems and provides
higher yields compared to mammalian cell expression (CHO cells).
Antibody fusion proteins require correct disulfide bridges and the
endoplasmic reticulum of yeast provides an oxidizing environment
like that of eukaryotic antibody producing cells. The multi-domain
structure of the bivalent immunotoxin requires a eukaryotic
expression system to properly fold this complex protein. Yet most
eukaryotes are sensitive to the effects of protein synthesis
inhibition upon expression of the immunotoxin. However, a budding
yeast, Pichia pastoris has a certain degree of tolerance to DT
(Neville et al., 1992; Woo et al., 2002) and yielded the
immunotoxin at a level of 40 mg/L in fermentor culture. The
immunotoxin was produced by fermentation of genetically engineered
Pichia pastoris (JW102, renamed from pJHW#2 (Woo et al., 2002)) via
the secretory route. As shown in Example 41, the present method
provides a yield of 120 mg/l after a 163 h induction period and the
purified yield is 90.8 mg/L. (see table 6).
After gene optimization to reduce the AT content of the DNA
sequence, secreted expression levels under the AOX1 promoter of
25-30 mg/L can be obtained in bioreactors after 24-44 hours of
induction. Pichia pastoris was sensitive to the toxic effects of
cytosolic expressed diphtheria toxin A chain which ADP ribosylates
elongation factor 2 (EF-2) leading to cessation of protein
synthesis. Toxicity to expression of A-dmDT390-bisFv by the
secretory route was indicated by a continuous fall in methanol
consumption after induction. A mixed feed of glycerol and methanol
was provided to the cells. Expression of the catalytic domain (A
chain) of DT in the cytosol is lethal to Pichia pastoris. When
cells bearing the construct A-dmDT390-BisFv (UCHT1) were induced by
methanol to express the immunotoxin, nearly 50% were killed after
24 hours (Woo et al., 2002). In contrast, when the same immunotoxin
was expressed in CHO cells that had been mutated to DT resistance,
no toxic effect was observed (Liu, et al., 2000; Thompson, et al.,
2001). In the cytosol of eukaryotes, the catalytic domain of DT
catalyzes ADP ribosylation of elongation factor 2 (EF-2), leading
to inhibition of protein synthesis and cell death (by protein
starvation and or apoptosis, Van Ness et al., 1980; Houchins,
2000). The sensitivity of the eukaryotic EF-2 to ADP-ribosylation
by these toxins lies in the structure of protein. EF-2 is a single
polypeptide chain of about 850 amino acids and is composed of two
domains. The N-terminal G domain is responsible for binding and
hydrolysis of GTP that promotes translation, and the C-terminal R
(or diphthamide) domain is thought to interact with the ribosome
(Kohno et al., 1986; Perentesis et al., 1992). The diphthamide
domain (FIG. 1) contains a histidine residue in a region of 22
residues that are well conserved in the EF-2 of all eukaryotes.
This conserved histidine is specifically modified
post-translationally to the derivative, diphthamide, which is the
unique target for ADP-ribosylation by DT (Van Ness et al., 1880).
In S. cerevisiae, the conserved histidine can be mutated and
substitutions with some other 2 amino acids yielded functional
EF-2s that were resistant to ADP-ribosylation (Phan et al., 1993;
Kimata and Kohno 1994). However, cells with EF-2 mutated at
diphthamide grew more slowly than those expressing wild-type EF-2.
In CHO cells, a single substitution of arginine for glycine, which
is another well conserved residue located at the 3rd position to
the C-terminal side of the diphthamide, also prevented the
formation of diphthamide (Kohno & Uchida, 1987; Foley et al.,
1992) and resulted in non-ADP-ribosylatable EF-2. This mutation had
the same effect on EF-2 of S. cerevisiae (Kimata et al., 1993). In
contrast to the mutation at diphthamide, the Gly to Arg mutation in
EF-2 did not affect cell growth of CHO and S. cerevisiae (Foley et
al., 1992; Kimata and Kohno 1994; Kimata et al., 1993).
In order to determine if the expression level of A-dmDT390-bisFv
could be further increased by rendering Pichia pastoris insensitive
to toxin, the EF-2 gene of Pichia pastoris has been mutated so that
the Gly at position 701 was changed to Arg, which has been shown to
prevent ADP-ribosylation of EF-2 in other organisms. The EF-2
mutagenesis required cloning of the gene, introduction of the in
vitro mutated sequence with a selection marker, URA3, to the genome
and PCR identification of mutated clones. The entire EF-2 gene of
Pichia pastoris has been cloned and sequenced. The coding sequence
of Pichia pastoris EF-2 is 2526 nucleotides coding for 842 amino
acids. The Pichia pastoris EF-2 is the same as the EF-2 of S.
cerevisiae and S. pombe in length and shares 88% and 78% of
identity in amino acid sequence with these two, respectively. In
contrast to these two yeasts, Pichia pastoris has only one copy of
the EF-2 gene that contains a short intron. Before the complete
sequence of EF-2 was known, different approaches were used to
mutate Pichia pastoris to obtain DT resistant strains. All these
efforts were unsuccessful due to the lack of robust selection.
Based on the EF-2 sequence obtained, a pBLURA-.DELTA.5'mutEF-2 was
constructed that targets Pichia pastoris EF-2 gene and introduces a
mutation of Gly 701 to Arg to the gene by homologous recombination.
The construct contains the 3' end 1028 nucleotides of EF-2 that has
been mutagenized in vitro to contain the amino acid substitution
and the auxotrophic marker URA3. A PCR detection method was also
developed for fast and accurate identification of mutant clones
after uracil selection. The targeted mutation strategy with
construct pBLURA-.DELTA.5'mutEF-2 allowed mutation of the EF-2 gene
of Pichia pastoris with about 40% of uracil positive clones being
found to contain the introduced mutations. EF-2 mutants were
developed with different auxotrophic markers, (specifically
mutEF2JC308 (ade1 arg4 his4), mutEF2JC303 (arg4 his4) and
mutEF2JC307 (his4)) and demonstrated that the Gly 701 to Arg
mutation in EF-2 confers resistance to the cytosolic expression of
DT A chain.
When EF-2 mutants were used to express A-dmDT390-bisFv under the
control of AOX1 promoter, they did not show the advantage over the
non-mutated expressing strain JW102 in the production of the
protein in shake-flask. However, in large-scale fermentation
culture under conditions adopted from those optimal for JW102, the
production of the mutant strain YYL#8-2 [mutEF2JC307-8(2)],
increased continuously for 96 hours and reached a level 1.46-fold
greater than the non-mutated JW102 strain. Cell growth and methanol
consumption rates of the mutant strain expressing A-dmDT390-bisFv
were the same as that of the non-expressing wild type strain.
Therefore it appeared that expression of A-dmDT390-bisFv was not
toxic to the mutant strain. The EF-2 mutants allowed expression of
A-dmDT390-bisFv under the control of the constitutive GAP promoter
(P.sub.GAP). In shake-flask culture, the production of
A-dmDT390-bisFv under P.sub.GAP was about 30% higher than that
under P.sub.AOX1. The increase in production under P.sub.GAP may be
more significant in fermentation cultures since fermentation allows
cells to grow to very high density.
In the Pichia pastoris expression system, most heterologous
proteins such as botulinum neurotoxin fragments for vaccine use
(Potter et al., 2000), hepatitis B surface antigen (Hardy et al.,
2000), gelatin (Werten et al., 1999), collagen (Nokelainen et al.,
2001), and insulin (Wang et al., 2001) were successfully expressed
and/or secreted by using a simple defined medium. The cytosolic
expression of the catalytic domain of DT causes protein synthesis
inhibition, leading to complete cell death in the defined medium,
but not in complex media (Liu et al., 2003). This finding indicates
that complex media play a role in attenuation of protein synthesis
inhibition that is caused by ADP-ribosylation of EF-2. A very low
production of the bivalent immunotoxin was observed in the defined
medium but not in a complex medium in shake flask culture.
Fermentation of Pichia pastoris for expression of heterologous
proteins had been developed on the basis of a defined medium but
use of complex media for expression of the bivalent immunotoxin in
a secreted form provides a higher level of production.
In the present large scale production of bivalent immunotoxin in
Pichia pastoris, lowering the induction temperature to 15.degree.
C. substantially improved the secretion of bioactive immunotoxin,
and thereby compensated for the limitation in Pichia pastoris
secretory capacity. In addition, the use of complex medium
containing yeast extract further enhanced immunotoxin secretion,
apparently by attenuating the toxic effects of the immunotoxin on
the Pichia pastoris host.
The expression level of the bivalent immunotoxin was improved by
4-fold in bioreactor culture compared to shake flask culture by
optimizing the fermentation conditions in Pichia pastoris as
follows: (1) use of Soytone Peptone and yeast extract based complex
medium, (2) use of methanol/glycerol mixed feed (4:1) to supplement
the energy source during methanol induction, (3) continuous feeding
of PMSF and yeast extract during induction, and (4) lowering
temperature to 15.degree. C. during methanol induction. The lowered
temperature resulted in a 2-fold increase in secretion relative to
using 23.degree. C. during methanol induction.
As noted above, a major problem in production of the bivalent
immunotoxin was reduction of methanol utilization during the
methanol induction phase. The reduction of methanol utilization
results from a reduction in the activity of the rate limiting
enzyme, alcohol oxidase (AOX1). This could be secondary to protein
synthesis inhibition by the bivalent immunotoxin reaching the
cytosol compartment through leakage from the secretory compartment
or by proton dependent translocation from the mildly acidic
secretory compartment (Arata et al., 2002). The fact that methanol
utilization is not affected by immunotoxin production in a Pichia
pastoris strain mutated to toxin resistance in the EF-2 gene (Liu
et al., 2003) indicates that toxin induced ADP-ribosylation is the
cause of the decreased AOX1 activity in strain JW102. However,
control of AOX1 level is balanced by both synthesis as well as
degradation, and degradative mechanisms could be augmented in
response to toxin mediated ADP-ribosylation. For reasons unknown,
yeast extract increased methanol utilization, though not to wild
type levels. In addition, low methanol utilization negatively
affected Pichia pastoris cell growth. This was corrected in the
present method by adding another carbon source, glycerol, and
continuous feeding of yeast extract during methanol induction.
These two corrections raised the methanol consumption to 80% of the
non-expressing strain.
To further compensate for Pichia pastoris protein synthesis
inhibition by the expressed immunotoxin, the fermentation
conditions were manipulated for full activation of alcohol oxidase
I (AOX1), the rate limiting enzyme for methanol metabolism
(Veenhuis et al., 1983). Since the immunotoxin gene was under the
control of the same strong promoter as the AOX1 gene, the
immunotoxin should be highly expressed. However, it has previously
been observed in the secretion of heterologous proteins that each
protein appears to have an optimal secretion level. Expression
beyond the optimal level (overexpression) of secreted heterologous
proteins can cause a reduction in secreted protein yields in
mammalian, insects and yeast cells (Bannister and Wittrup, 2000;
Liebman et al., 1999; Liu et al., 2003; Pendse et al., 1992). In
order to determine whether the bivalent immunotoxin was being
overexpressed in Pichia pastoris, the induction temperature was
lowered during methanol induction. Since most cellular activities
including protein synthesis are decreased at low temperature,
lowering induction temperature should decrease the synthetic rate
of the bivalent immunotoxin. Any resulting change in secretion rate
was judged. Bivalent immunotoxin expression was increased at low
induction temperatures, reaching a maximum at 17.5.degree. C., and
secretion of bioactive immunotoxin reached a maximum at 15.degree.
C., in spite of the fact that methanol consumption rate at
15.degree. C. fell to 75% of its 23.degree. C. value. Because
continuous feeding of PMSF and yeast extract during induction
effectively inhibited protease activity in supernatants, it appears
unlikely that a reduction in protease activity with lower induction
temperature accounts for the nearly 2-fold increase in bivalent
immunotoxin secretion seen at 15.degree. C. The limitation in
Pichia pastoris secretion of bivalent immunotoxin previously
described may actually represent an overexpression at 23.degree. C.
that is reduced at 15.degree. C. achieving a better balance of
input and output within the secretory compartment.
In short, the immunotoxin was produced in Pichia pastoris (JW102)
via the secretory route under control of the AOX1 promoter in the
fermentor using methanol as a carbon source. There were two major
impediments to efficient immunotoxin production, the toxicity of
the immunotoxin towards Pichia pastoris and the limited secretory
capacity of Pichia pastoris for the immunotoxin. The toxicity
towards Pichia pastoris resulted in a decrease in the metabolic
rate of methanol consumption, a cell growth rate reduction and very
low productivity in a defined medium during methanol induction.
These problems were overcome by (1) using an enzymatic digest of
soy protein (e.g., Soytone peptone) and yeast extract based complex
medium, (2) using methanol/glycerol mixed feed (4:1) to supplement
energy source during methanol induction, and (3) continuously
feeding PMSF and yeast extract during methanol induction. Lowering
the induction temperature to 15.degree. C. improved secreted
immunotoxin yield by almost 2-fold, up to 40 mg/L (at 67 hours
induction) compared to secretion at a induction temperature of
23.degree. C., even though methanol consumption was reduced. In
addition, with the use of the present method, the fraction of
immunotoxin present as biologically inactive oligomeric forms was
decreased.
Also provided by the invention is a method of purifying a
non-glycosylated immunotoxin comprising (a) loading a solution
containing the non-glycosylated immunotoxin onto a hydrophobic
interaction column; (b) obtaining a first non-glycosylated
immunotoxin containing eluant from the hydrophobic interaction
column; (c) loading the non-glycosylated immunotoxin containing
eluant from step (b) onto an anion exchange column; (d) obtaining a
second non-glycosylated immunotoxin containing eluant from the
anion exchange column by eluting the non-glycosylated immunotoxin
with a sodium borate solution; (e) diluting the concentration of
sodium borate in the second non-glycosylated immunotoxin containing
eluant from step (d) to about 50 mM or less; (f) concentrating the
diluted non-glycosylated immunotoxin containing eluant from step
(e) over an anion exchange column; and (g) obtaining a purified
non-glycosylated immunotoxin from the anion exchange column.
Optionally, the method further comprises washing the anion exchange
column with about 25 mM sodium borate solution prior to eluting
with the sodium borate solution. Preferrably the non-glycosylated
immunotoxin being purified is expressed by the methods taught
herein.
The concentration of the sodium borate solution in step (d) of the
purification method is between about 25 mM and about 200 mM, and
preferably is between about 75 mM and about 100 mM. For example,
the concentration of sodium borate in step (e) can be about 20
mM.
A major problem encountered in the large scale purification of the
bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G.sub.4S), from
Pichia pastoris supernatants is the presence of host glycoproteins
exhibiting similar charge, size and hydrophobicity characteristics.
This problem was overcome by employing borate anion exchange
chromatography. Borate anion has an affinity for carbohydrates and
imparts negative charges to these structures. At a concentration of
sodium borate between 50 and 100 mM, the non-glycosylated
immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but
glycoproteins, including aggregates related to the immunotoxin, did
bind. By using this property of the immunotoxin in the presence of
sodium borate, a 3-step purification procedure was developed: (1)
Butyl 650M hydrophobic interaction chromatography, (2) Poros 50 HQ
anion exchange chromatography in the presence of borate, and (3) Q
anion exchange chromatography. This procedure has several
advantages: (1) it is a relatively simple process without any
dialysis or diafiltration step; (2) it exhibits good repeatability;
(3) the final yield is over 50%; and (4) the final purity is over
98%. Previously, boronic acid resins have been used to separate
glycoproteins from proteins. However, combining borate anion with
conventional anion exchange resins accomplishes separation of the
immunotoxin from glycoproteins, and eliminates the need to evaluate
non-standard resins with respect to good manufacturing practice
guidelines. Thus, borate anion exchange chromatography was used for
separation of the immunotoxin from Pichia pastoris
glycoproteins.
The immunotoxin is functionally active only in its monomeric form.
However the supernatant harvested from the fermentation run
contained monomeric, dimeric and higher oligomeric forms of the
immunotoxin as well as Pichia pastoris proteins. Among these Pichia
pastoris proteins, a glycoprotein species of approximately 45 kDa
was present in dimeric form (.about.90 kDa). The dimeric and higher
oligomeric forms of the immunotoxin were relatively easy to
separate by the use of conventional hydrophobic interaction
chromatography and anion exchange chromatography. However, it was
difficult to isolate the pure monomeric immunotoxin because the 45
kDa glycoproteins were very similar to the monomeric immunotoxin in
size, hydrophobicity, and isoelectric point.
Previously, immobilized phenylboronate resins have been used for
separation of glycoproteins from proteins (Myohanen et al., 1981;
Bouritis et al., 1981; Williams et al., 1981; Zanette et al.,
2003). These immobilized resins bind and selectively retard
glycoproteins depending on pH, presence of sugar, type of sugar,
concentration of sugar and buffer species. Borate anion exchange
chromatography is used rather than the immobilized phenylboronate
affinity chromatography for separation of the immunotoxin from the
45 kDa glycoprotiens, because of poor separation capability of
phenylboronate resin. Borate forms complexes with sugar residues
having vicinal cis-hydroxyl groups (Boeseken, 1949) and these
complexes are reversible (Weigel, 1963). Reversible complex
formation of borate with carbohydrate on glycoproteins resulted in
an increased negative charge of the glycoproteins. This property
allowed separation of non-glycoproteins and glycoproteins on anion
exchange chromatography (Nomoto et al., 1982; Nomoto and Inoue,
1983).
In the separation of the immunotoxin from glycoproteins, borate
anion exchange chromatography had different binding characteristics
from phenylboronate affinity chromatography. In phenyloboronate
affinity chromatography, glycoproteins as well as the immunotoxin
were bound under the condition of low ionic strength and they were
co-eluted by either 0-100 mM sodium borate gradient or 0-50 mM
sorbitol gradient, indicating that the immunotoxin physically
interacts with at least one of the bound glycoproteins, or
interacts with the phenylboronate column through an alternate
mechanism. The fact that purified bivalent immunotoxin also bound
to the phenylboronate column indicates binding through an alternate
mechanism.
In previous purification methods utilizing shake flask culture, a
2-step procedure was employed which involved DEAE anion exchange
chromatography and Protein L affinity chromatography for
purification of the immunotoxin (Woo et al., 2002). However, the
supernatants of high density fermentor cultures of Pichia pastoris
contain materials that severely reduce the capacity of anion
exchange resins. In addition, the Protein L affinity step required
excessive column size, was expensive and was not available under
Good Manufacturing Practices (GMP) certification. Consequently,
alternative procedures were developed. Hydrophobic interaction
chromatography using Butyl 650M worked well as a capture step but
also concentrated P. pastoris glycoproteins having similar charge,
size and hydrophobicity as the immunotoxin. Concanavalin A affinity
resin was promising for glycoprotein removal, but bleeding of
potentially toxic concanavalin A from the resin resulted in
unacceptable contamination of the final product.
The anion exchange column may be but is not limited to an anion
acrylic, anion agarose, anion cellulose, anion dextran or anion
polystyrene. The preferred anion exchange columns are a Poros HQ 50
and a Q anion exchange column. By using the Poros 50 HQ borate
exchange chromatography in the present invention, substantial
purification of the monomeric form of the immunotoxin was obtained,
even though the immunotoxin in the eluted faction was diluted.
Thus, a subsequent concentration step by Q anion exchange
chromatography was used.
The hydrophobic interaction column may be but is not limited to a
Phenyl-SEPHAROSE.RTM. CL-4B, Octyl Agarose, Phenyl-Sepharose 6 Fast
Flow, Phenyl-Agarose, Phenyl-Sepharose 6 Fast Flow, Octyl-Sepharose
4 Fast Flow, Butyl Sepharose.TM. 4 Fast Flow, Octyl Agarose,
Phenyl-Agarose, Hydrophobic chromatography media-monoamino MAA-8,
Hexyl-Agarose, Dodecyl-Agarose, Hexyl-Agarose,
4-Phenylbutylamine-Agarose, Ethyl-Agarose, Matrix, Butyl-Agarose,
Propyl Agarose, Afinity chromatography media AAF-8, Octyl Agarose,
Butyl-Agarose, Decyl-Agarose, Phenyl-Agarose, Methyl Matrix:
Ceramic HyperD F Hydrogel Composite, Octyl Agarose, Trityl-Agarose,
Q Sepharose, Ether 650, Phenyl 650, Butyl 650 or Hexyl 650. The
preferred hydrophobic interaction column is a Butyl 650M
hydrophobic interaction column.
The present borate anion exchange chromatography is useful for the
purification of other Pichia pastoris expressed proteins. Pichia
pastoris is being increasingly used as an expression system for
therapeutic recombinant proteins (Cereghino et al., 2002). Many of
these recombinant proteins have their glycosylation sites removed
due to the profound differences in glycosylation patterns between
Pichia pastoris and humans. These recombinant proteins are then
amenable to purification using borate anion exchange
chromatography.
It is contemplated that any buffer, flow rate, and column size may
be used that would successfully effect elution of the immunotoxin
in a more pure state than the immunotoxin was loaded upon the
column.
An immunotoxin used in the present invention comprises a mutant
toxin moiety (e.g., DT toxin) linked to an antibody moiety
(targeting moiety). Toxins that may be used include but are not
limited to diphtheria toxin, ricin toxin, and pseudomonas exotoxin.
The antibody moiety is preferably a single chain (sc) variable
region.
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, protein chemistry and immunology,
which are within the skill of the art. Such techniques are
explained fully in the literature, including Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor
Laboratory Press, 1989); DNA CLONING, Vol. I and II, D. N Glover
ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M. J. Gait ed.
(IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B. D. Hames & S.
J. Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION,
B. D. Hames & S. J. Higgins eds., (IRL Press, 1984); ANIMAL
CELL CULTURE, R. I. Freshney ed. (IRL Press, 1986); IMMOBILIZED
CELLS AND ENZYMES, K. Mosbach (IRL Press, 1986); B. Perbal, A
PRACTICAL GUIDE TO MOLECULAR CLONING, Wiley (1984); the series,
METHODS IN ENZYMOLOGY, Academic Press, Inc.; GENE TRANSFER VECTORS
FOR MAMMALIAN CELLS, J. H. Miller and M. P. Calos eds. (Cold Spring
Harbor Laboratory, 1987); METHODS IN ENZYMOLOGY, Vol. 154 and 155,
Wu and Grossman, eds., and Wu, ed., respectively (Academic Press,
1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, R. J.
Mayer and J. H. Walker, eds. (Academic Press London, Harcourt Brace
U.S., 1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed.
(Springer-Verlag, N.Y. (1987), and HANDBOOK OF EXPERIMENTAL
IMMUNOLOGY, Vol. I-IV, D. M. Weir et al., (Blackwell Scientific
Publications, 1986); Kitts et al., Biotechniques 14:810-817 (1993);
Munemitsu et al., Mol. and Cell. Biol. 10:5977-5982 (1990).
The present invention utilizes a nucleic acid encoding a diphtheria
toxin-containing fusion protein, wherein the nucleic acid can be
expressed by a yeast cell. The nucleic acid capable of being
expressed by yeast, comprises a modified native diphtheria-encoding
sequence. To promote expression of the nucleic acids of the present
invention by yeast cells, regions of the nucleic acid rich in A and
T nucleotides are modified to permit expression of the encoded
immunotoxin fusion protein by yeast. For example, such
modifications permit expression by Pichia pastoris. The
modifications are designed to inhibit polyadenylation signals
and/or to decrease early termination of RNA transcription. More
specifically, one or more AT rich regions of the native
diphtheria-encoding sequence are modified to reduce the AT content.
The AT rich regions include regions of at least 150 contiguous
nucleotides having an AT content of at least 60% or regions of at
least 90 contiguous nucleotides having an AT content of at least
65%, and the AT content of the AT rich regions is preferably
reduced to 55% or lower. The AT rich regions also include regions
of at least 150 contiguous nucleotides having an AT content of at
least 63% or regions of at least 90 contiguous nucleotides having
an AT content of at least 68%, and the AT content of the AT rich
regions is reduced to 55% or lower. The native diphtheria-encoding
sequence preferably is further modified to encode a diphtheria
toxin truncated at its C-terminal. Furthermore, the native
diphtheria-encoding sequence preferably is further modified to
encode one or more amino acids prior to the amino terminal glycine
residue of the native diphtheria toxin. Furthermore, the native
diphtheria-encoding sequence preferably is further modified to
encode the alpha mating factor signal peptide or a portion
thereof.
The immunotoxin of the present invention may be expressed in and
purified from various organisms. These organisms include yeast such
as Pichia pastoris or Saccharomyces cerevisiae, bacteria such as
Escherichia coli, mammalian cells such as Chinese hamster ovary
cells or baculovirus infected insect cells. There are several
advantages to yeast expression systems, which include, for example,
Saccharomyces cerevisiae and Pichia pastoris. First, evidence
exists that proteins produced in a yeast secretion systems exhibit
correct disulfide pairing. Second, efficient large scale production
can be carried out using yeast expression systems. The
Saccharomyces cerevisiae pre-pro-alpha mating factor leader region
can be used to direct protein secretion from yeast (Brake, et al.
(82)). The leader region of pre-pro-alpha mating factor contains a
signal peptide and a pro-segment which includes a recognition
sequence for a yeast protease encoded by the KEX2 gene: this enzyme
cleaves the precursor protein on the carboxyl side of a Lys-Arg
dipeptide cleavage signal sequence. The nucleic acid coding
sequence can be fused in-frame to the pre-pro-alpha mating factor
leader region. This construct can be put under the control of a
strong transcription promoter, such as the alcohol dehydrogenase I
promoter, alcohol oxidase I promoter, a glycolytic promoter, or a
promoter for the galactose utilization pathway. The nucleic acid
coding sequence is followed by a translation termination codon
which is followed by transcription termination signals.
Alternatively, the nucleic acid coding sequences can be fused to a
second protein coding sequence, such as Sj26 or beta-galactosidase,
used to facilitate purification of the fusion protein by affinity
chromatography. The insertion of protease cleavage sites to
separate the components of the fusion protein is applicable to
constructs used for expression in yeast.
Diphtheria toxin is toxic to yeast when the toxin A chain is
synthesized within the cytosol compartment without a secretory
signal (Parentesis et al., 1988). This toxin-catalyzed activity is
specific for EF-2 and occurs at a unique post-translational
histidine residue at the position 699, found in a conserved amino
acid sequence in the EF-2 of all eukaryotes. A change of glycine to
arginine residue at the position 701 in yeast EF-2 results in
resistance to DT.
In an alternative purification method, (Ala)dmDT390-bisFv(UCHT1)
was produced in the Pichia medium at a level of 5 mg/ml whether or
not the mutant EF-2 gene was present. There is an extremely tight
coupling between the presence of the alpha-mating factor signal
sequence and the compartmentalization of (Ala)dmDT390-bisFv(UCHT1)
into the secretory pathway and away from the EF-2 toxin substrate
in the cytosol compartment, since one molecule of toxin in the
cytosol can inactivate 99% of the EF-2 in 24 hours. Producing
(Ala)dmDT390-bisFv(UCHT1) in Pichia utilizing the alpha-mating
factor signal sequence without mutating the Pichia to toxin
resistance provided a successful outcome. Another combination of a
yeast produced toxin (ricin A chain) and signal sequence, Kar2,
resulted in death of the producing cells (Simpson et al., 1999
(80)). It is possible that, at higher gene dosages of immunotoxin
fusion protein in Pichia, mEF-2 may confer a benefit in production.
A further advantage of yeast over mammalian cells for immunotoxin
fusion protein production is the fact that intact yeast are highly
resistant to diphtheria toxin present in the external medium to
levels as high as 3.3.times.10-6 M. Evidently the yeast capsule
prevents retrograde transport of toxin back into the cytosol
compartment as occurs in mammalian cells and in yeast spheroplasts
(Chen et al. 1985).
The invention may utilize a cell comprising a nucleic acid that
encodes the immunotoxin fusion protein. The cell can be a
prokaryotic cell, including, for example, a bacterial cell. More
particularly, the bacterial cell can be an E. coli cell.
Alternatively, the cell can be a eukaryotic cell, including, for
example, a Chinese hamster ovary (CHO) cell (including for example,
the DT resistance CHO-K1 RE 1.22c cell line, as selected by
Moehring & Moehring), myeloma cell, a Pichia cell, or an insect
cell. The immunotoxin fusion protein coding sequence can be
introduced into a Chinese hamster ovary (CHO) cell line, for
example, using a methotrexate resistance-encoding vector, or other
cell lines using suitable selection markers. Presence of the vector
DNA in transformed cells can be confirmed by Southern blot
analysis. Production of RNA corresponding to the insert coding
sequence can be confirmed by Northern blot analysis. A number of
other suitable host cell lines have been developed and include
myeloma cell lines, fibroblast cell lines, and a variety of tumor
cell lines such as melanoma cell lines. Expression vectors for
these cells can include expression control sequences, such as an
origin of replication, a promoter, an enhancer, and necessary
information processing sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites, and transcriptional terminator
sequences. Preferred expression control sequences are promoters
derived from immunoglobulin genes, SV40, Adenovirus, Bovine
Papilloma Virus, etc. The vectors containing the nucleic acid
segments of interest can be transferred into the host cell by
well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transformation is commonly
utilized for prokaryotic cells, whereas calcium phosphate, DEAE
dextran, or lipofectin mediated transfection or electroporation may
be used for other cellular hosts.
The nucleic acids used in the present invention can be operatively
linked to one or more of the functional elements that direct and
regulate transcription of the inserted nucleic acid and the nucleic
acid can be expressed. For example, a nucleic acid can be
operatively linked to a bacterial or phage promoter and used to
direct the transcription of the nucleic acid in vitro.
A mutant strain of Pichia pastoris is provided. The mutant strain
comprises a mutation in at least one gene encoding elongation
factor 2 (EF2). This mutation comprises a Gly to Arg replacement at
a position two residues to the carboxyl side of the modified
histidine residue diphthamide. In this manner, the strain is made
resistant to the toxic ADP-ribosyating activity of diphtheria
toxin.
A method of expressing a diphtheria toxin protein moiety is
provided. Such a method of the invention comprises transfecting a
mutated Pichia pastoris cell of the invention with a vector
comprising a toxin protein-encoding nucleic acid under conditions
that permit expression of the protein-encoding nucleic acid in the
cell. The conditions are those used for Pichia pastoris cells and
can be optimized for the particular system.
The antibody moiety preferably routes by the anti-CD3 pathway or
other T cell epitope pathway. The immunotoxin can be monovalent,
but bivalent antibody moieties are presently preferred since they
have been found to enhance cell killing by about 15 fold. It is
contemplated that any number of chemical coupling or recombinant
DNA methods can be used to generate an immunotoxin of the
invention. Thus, reference to a fusion toxin or a coupled toxin is
not necessarily limiting. The immunotoxin can be a fusion protein
produced recombinantly. The immunotoxin can be made by chemical
thioether linkage at unique sites of a recombinantly produced
bivalent antibody (targeting moiety) and a recombinantly produced
mutant toxin moiety. The targeting moiety of the immunotoxin can
comprise the human .mu.CH2, .mu.CH3 and .mu.CH4 regions and VL and
VH regions from murine Ig antibodies. These regions can be from the
antibody UCHT1 so that the antibody moiety is scUCHT1, which is a
single chain CD3 antibody having human .mu.CH2, .mu.CH3 and .mu.CH4
regions and mouse variable regions as shown in the figures.
Numerous DT mutant toxin moieties are contemplated, including for
example, DT390 and DT389, with a variety of mutations or as the
wild type toxin moiety.
The toxin moiety retains its toxic function, and membrane
translocation function to the cytosol in full amounts. The loss in
binding function located in the receptor binding domain of the
protein diminishes systemic toxicity by reducing binding to
non-target cells. Thus, the immunotoxin can be safely administered.
The routing function normally supplied by the toxin binding
function is supplied by the targeting antibody anti-CD3. The
essential routing pathway is (1) localization to coated pits for
endocytosis, (2) escape from lysosomal routing, and (3) return to
the plasma membrane.
Any antibody that can route in this manner will be effective with
the toxin moiety, irrespective of the epitope to which the antibody
is directed, provided that the toxin achieves adequate proteolytic
processing along this route. Adequate processing can be determined
by the level of cell killing.
When antibodies dissociate from their receptors due to changes in
receptor configuration induced in certain receptors as a
consequence of endosomal acidification, they enter the lysosomal
pathway. This can be prevented or minimized by directing the
antibody towards an ecto-domain epitope on the same receptor which
is closer to the plasma membranes (Ruud, et al. (1989) Scand. J.
Immunol. 29:299; Herz et al. (1990) J. Biol. Chem. 265:21355).
The mutant DT toxin moiety can be a truncated mutant, such as
DT390, DT389 or DT383, or other truncated mutants, with and without
point mutations or substitutions, as well as a full length toxin
with point mutations, such as DTM1, or CRM9 (cloned in C.
ulcerans), scUCHT1 fusion proteins with DTM1 and DT483, DT390 and
DT389, and have been cloned and expressed in E. coli. The antibody
moiety can be scUCHT1 or other anti-CD3 or anti-T cell antibody
having the routing and other characteristics described in detail
herein. Thus, one example of an immunotoxin for use in the present
methods comprises the fusion protein immunotoxin UCHT1 (or a
fragment thereof)-DT390.
There is a consensus sequence for glycosylation (NXS/T (SEQ ID NO:
19)) that may be removed or inserted to control glycosylation.
Glycosylation occurs in all eukaryotes, e.g. Pichia pastoris.
There are numerous variants of the immunotoxins. Protein variants
and derivatives are well understood to those of skill in the art
and can involve amino acid sequence modifications. For example,
amino acid sequence modifications typically fall into one or more
of three classes: substitutional, insertional or deletional
variants. Insertions include amino and/or carboxyl terminal fusions
as well as intrasequence insertions of single or multiple amino
acid residues. Insertions ordinarily will be smaller insertions
than those of amino or carboxyl terminal fusions, for example, on
the order of one to four residues. Immunogenic fusion protein
derivatives, such as those described in the examples, are made by
fusing a polypeptide sufficiently large to confer immunogenicity to
the target sequence by cross-linking in vitro or by recombinant
cell culture transformed with DNA encoding the fusion. Deletions
are characterized by the removal of one or more amino acid residues
from the protein sequence. Typically, no more than about from 2 to
6 residues are deleted at any one site within the protein molecule.
These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing
DNA encoding the variant, and thereafter expressing the DNA in
recombinant cell culture. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example M13 primer mutagenesis and PCR mutagenesis.
Amino acid substitutions are typically of single residues, but can
occur at a number of different locations at once; insertions
usually will be on the order of about from 1 to 10 amino acid
residues; and deletions will range about from 1 to 30 residues.
Deletions or insertions preferably are made in adjacent pairs, i.e.
a deletion of 2 residues or insertion of 2 residues. Substitutions,
deletions, insertions or any combination thereof may be combined to
arrive at a final construct. The mutations must not place the
sequence out of reading frame and preferably will not create
complementary regions that could produce secondary mRNA structure.
Substitutional variants are those in which at least one residue has
been removed and a different residue inserted in its place. Such
substitutions generally are made in accordance with the following
are referred to as conservative substitutions.
TABLE-US-00001 Amino Acid Abbreviations Amino Acid Abbreviations
Alanine Ala, A Allosoleucine AIle Arginine Arg, R Asparagine Asn, N
aspartic acid Asp, D Cysteine Cys, C glutamic acid Glu, E Glutamine
Gln, K Glycine Gly, G Histidine His, H Isolelucine Ile, I Leucine
Leu, L Lysine Lys, K Phenylalanine Phe, F Proline Pro, P
Pyroglutamic acid PGlu Serine Ser, S Threonine Thr, T Tyrosine Tyr,
Y Tryptophan Trp, W Valine Val, V
Amino Acid Substitutions
Original Residue Exemplary Conservative Substitutions, others are
known in the art.
TABLE-US-00002 Ala ser Arg lys, gln Asn gln, his Asp glu Cys ser
Gln asn, lys Glu asp Gly pro His asn, gln Ile leu, val Leu ile, val
Lys arg, gln; Met leu, ile Phe met, leu, tyr Ser thr Thr ser Trp
tyr Tyr trp, phe Val ile, leu
Substantial changes in function or immunological identity are made
by selecting substitutions that are less conservative than those in
the amino acid substitution table, i.e., selecting residues that
differ more significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site or
(c) the bulk of the side chain. The substitutions that in general
are expected to produce the greatest changes in the protein
properties will be those in which (a) a hydrophilic residue, e.g.
seryl or threonyl, is substituted for (or by) a hydrophobic
residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b)
a cysteine or proline is substituted for (or by) any other residue;
(c) a residue having an electropositive side chain, e.g., lysyl,
arginyl, or histidyl, is substituted for (or by) an electronegative
residue, e.g., glutamyl or aspartyl; or (d) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by)
one not having a side chain, e.g., glycine, in this case, (e) by
increasing the number of sites for sulfation and/or
glycosylation.
For example, the replacement of one amino acid residue with another
that is biologically and/or chemically similar is known to those
skilled in the art as a conservative substitution. For example, a
conservative substitution would be replacing one hydrophobic
residue for another, or one polar residue for another. The
substitutions include combinations such as, for example, Gly, Ala;
Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,
Tyr. Such conservatively substituted variations of each explicitly
disclosed sequence are included within the mosaic polypeptides
provided herein.
Substitutional or deletional mutagenesis can be employed to insert
sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser
or Thr). Deletions of cysteine or other labile residues also may be
desirable. Deletions or substitutions of potential proteolysis
sites, e.g. Arg, are accomplished for example by deleting one of
the basic residues or substituting one by glutaminyl or histidyl
residues.
Certain post-translational derivatizations are the result of the
action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human or nucleated cells) may also contain
sequences necessary for the termination of transcription which may
affect mRNA expression. These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding tissue factor protein. The 3' untranslated regions also
include transcription termination sites. It is preferred that the
transcription unit also contain a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that homologous
polyadenylation signals be used in the transgene constructs. In
certain transcription units, the polyadenylation region is derived
from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain
other standard sequences alone or in combination with the above
sequences improve expression from, or stability of, the
construct.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Example 1
Transformation with a Mutagenizing Oligonucleotide
The oligomer of 56 nucleotides (see List of Primers) contains two
point mutations to change amino acid 701 from glycine to arginine.
A mutagenizing oligo (56 mer, 100 ug) was co-transformed into the
GS200 (Mut+, His-, Arg-) strain with an ARG4 DNA fragment. The ARG4
gene with promoter was taken from plasmid PMY30 (supplied by Prof.
Jim Cregg, Keck Graduate Institute of Applied Life Sciences,
Claremont, Calif. 91711) by Sph I and EcoR V. Approximately 1000
transformants were obtained. To screen for mutated clones having
the correct mutations, diagnostic PCR with primers mdb1EF-2 and
2253EF-2C was employed. The mutation-detecting primer (mdb1EF-2)
can distinguish a difference in DNA sequence between the normal
gene and the mutated gene at amino acid 701. For the normal gene, a
PCR product could not be produced because 2 nucleotides at the 3'
end were not matched with the DNA sequence of the normal gene,
preventing extension by the Taq polymerase. For the mutated gene,
the primer could anneal perfectly, so Taq polymerase could produce
a PCR product. More than 1000 colonies were screened by this PCR
method but no mutated colony was identified. (In the above PCR
assay, amino acid 701 mutated EF-2 from S. cerevisiae served as a
positive control. This mutated gene had been made previously with
the intent of introducing it into Pichia pastoris. However, Pichia
pastoris thus transformed had a very slow growth rate and produced
the protein of interest at low levels.)
Transformation was performed with a partial DNA fragment containing
the conserved region of the EF-2 gene and a mutation on amino acid
701. The partial sequence of Pichia pastoris EF-2 (positions 1717
to 2289, FIG. 3 (a) (b) (c) (d)) was mutated in vitro to change the
amino acid 701 from glycine to arginine (FIG. 1) and then
co-transformed into the GS200 strain with the ARG4 gene fragment.
More than 2000 Arg4 positive transformants were obtained and
screened them for the EF-2 mutation by diagnostic PCR with primers
mdb2EF-2 and 2253EF-2C. The mutation was not observed.
List of Primers
Primers Derived from S. cerevisiae EF-2:
TABLE-US-00003 (SEQ ID NO: 20) 5' primer: TTG GTT ATT GAC CAA ACT
AAG GCT GTCCAA (SEQ ID NO: 21) 3' primer: ACC TCT CTT CTT GTT TAA
GAC GGA GTA GAT
Primers Used in Cloning and Mutation of Pichia pastoris EF-2
TABLE-US-00004 dT.sub.22-Not: (SEQ ID NO: 22) 5'-CTT GCT TTT GCG
GCC GCT TTT TTT TTT TTT TTT TTT TTT EF-2C.sub.2: (SEQ ID NO: 23)
5'-G ATA AGA ATG CGG CCG CCA TTT CTT GGT CTT TGG GTT GAA G
EF-2C.sub.1: (SEQ ID NO: 24) 5'-GAT AAG AAT GCG GCC GCC AAC TTA GTT
GTT GAG GAG TCT AAG 5'EF-2: (SEQ ID NO: 25) 5'-ATA GCT AGC ACT TTG
AAG TTC TTA ATT TTG TTC CTC 3'EF-2C: (SEQ ID NO: 26) 5'-ATA AGA ATG
CGG CCG CAA GTT AAT GAA ACA TTA AGC TTA CAA C wEF-2: (SEQ ID NO:
27) 5'-G AAT GAC TTG TCC TCC ACC mEF-2: (SEQ ID NO: 28) 5'-G AAT
GAC TTG TCC TCC GCG G EF-1426: (SEQ ID NO: 29) 5'-CAA CTA GCT AGC
GCT CAC AAC ATG AAG GTC ATG AAA TTC EF-1318: (SEQ ID NO: 30) 5'-AGA
ACC GTC GAG CCT ATT GAC GAT
Mutagenizing Oligo:
TABLE-US-00005 (SEQ ID NO: 31) 5'-CC CTG CAC GCC GAT GCT ATC CAC
AGA AGA GGA GGA CAA GTC ATT CCA ACC ATG AAG mdb1EF-2: (SEQ ID NO:
32) 5'-GCC GAT GCT ATC CAC AGA AGA mbb2EF-2: (SEQ ID NO: 33) GCC
GAT GCT ATC CAC CGC CGC 2253EF-2C: (SEQ ID NO: 34) TCT CTT CTT GTT
CAA AAC AGA GTA GAT ACC
Example 2
Spheroplast Transformation with the Partial Fragment of Mutated EF2
and ARG4 Fragment
In the methods of Example 1, there was no selection step against
wild type DT. A double transformation was thus employed. First, the
mutated EF-2 fragment was transformed into the GS200 strain by
electroporation. Then, electroporated cells were cultivated
overnight to allow the expression of mutated EF-2 inside cell.
Cultivated cells were used for making spheroplasts. The resulting
spheroplasts were treated with wild type DT (200 .mu.g/ml) and ARG4
fragment (10 .mu.g) for 1 hour and transformed by CaCl.sub.2 and
PEG. Only a few transformants of normal colony size were obtained
and there was no mutated strain. In addition, there were 100 or
more micro-colonies obtained. 100 of these were screened but the
mutated strain was not detected.
Example 3
Cloning and Sequencing of EF-2 Gene from Pichia pastoris
Prior to the cloning of the full sequence of the Pichia pastoris
EF-2 gene, a partial sequence had been obtained. Initially, the
conserved R domain of Pichia pastoris EF-2 was amplified from the
genomic DNA using two primers derived from the same region of S.
cerevisiae EF-2 (Perentesis et al., 1992). The 5' primer contained
the sequence from position 1933 to 1962 of S. cerevisiae EF-2,
whereas the 3' primer was complementary to the region of 2227 to
2256. The sequence of 324 nucleotides was then extended towards the
5' end to position 284 and the 3' end to position 2289 in the
coding region of Pichia pastoris EF-2 gene. The extended sequence
was later found to contain several mistakes. To clone the entire
Pichia pastoris EF-2 gene, two species of cDNA were first
synthesized separately from EF-2 mRNA with two different primers.
Primer dT22-Not contains a run of 22 T residues complementary to
the 3'polyA tail of the mRNA and the recognition sequence for
restriction enzyme Not 1. Primer EF-2C2 has 25 nucleotides
complementary to nucleotide positions 747 to 771 (FIG. 3(a) (b) (c)
(d)) of the Pichia pastoris EF-2 coding sequence. After cDNA
synthesis, a homopolymeric track of A residues was added to the 3'
end of the cDNA extended from primer C2 by homopolymeric tailing
(Sambrook et al., 1989). The 5' end sequence of EF-2 was amplified
by PCR from the modified cDNA with EF-2C2 and dT22-Not primers,
whereas the 3' end sequence was from the cDNA synthesized from
primers dT22-Not and EF-2C1, which contains 27 nucleotides
corresponding to the positions 1927 to 1953. The PCR products
representing the 5' end and 3' end sequences of Pichia pastoris
EF-2 were then separately cloned to pCR2.1-TOPO vector
(Invitrogen).
Five independent clones containing 5' sequence of EF-2 Pichia
pastoris were selected for sequencing. They were first sequenced
with M13 reverse and M13 forward primers located in the vector
close to the up and down streams of the insert respectively, and
then with an internal prime complementary to the positions 349 to
384 of EF-2 coding sequence. Among the 5 clones, three had
identical sequences, one had two different nucleotides at two
different internal locations, and the other one had another
different internal nucleotides at a different location. These
different nucleotides were likely produced by the cloning
procedures since none of these different nucleotides were present
in the clone derived from genomic DNA. At the 5' end, all five
clones also had 57 to 69 nucleotides of the same sequence before
the first ATG codon. The largest open reading frame (ORF) of the 5'
end sequence starting from the first AUG is 747 nucleotides and the
deduced amino acid sequence (249 aa) shares 90% identity with the
first 249 aa at the N-terminus of S. cerevisiae EF-2. All four
clones containing the 3' end sequence of the EF-2 sequence had the
same sequence of 675 nucleotides followed by a homopolymeric A
track. The largest ORF is 603 nucleotides ended at stop codon TAA,
which is 72 nucleotides up stream of the poly-A track. The deduced
amino acid sequence (201 aa) shares 85% identity with the last 201
aa at the C-terminus of S. cerevisiae EF-2. Having obtained both
the 5' and 3' end sequences of Pichia pastoris EF-2, two primers
were designed to amplify the entire the EF-2 gene from the genomic
DNA of Pichia pastoris. Primer 5'EF-2 is derived from the 5'
non-coding region and contains the sequence from positions 28 to 54
relative to the ATG initiation codon. Primer 3'EF-2C contains 27
nucleotides complementary to positions 2523 to 2549 at the 3' end.
After PCR amplification with Pfu polymerase (Stratagene), the PCR
products of EF-2 gene were treated with Taq polymerase to have the
3'A-overhangings added (Instruction manual for original TA cloning
kit, Invitrogen) and then inserted into the TA cloning vector
pCR2.1-TOPO. Ten clones were picked, and the restriction enzyme
analysis of plasmid DNA isolated from these clones indicated that
they all had the same insert. DNA sequencing was performed first
with M13 reverse and M13 forward primers and then advanced step by
step towards the opposite ends with primers derived from the
sequences obtained from the previous steps. Eight clones were
completely sequenced, and found to be identical. The 3' end
sequence obtained from the genomic DNA is identical to that from
the mRNA. However, compared to the 5'sequence of mRNA, the sequence
from the genomic DNA has an insertion of 77 nucleotides in the
codon immediately next to the initiation site of the EF-2 ORF (FIG.
2). The insertion has the sequence GTATGT . . . CACTAAC . . . TAG
(SEQ ID NO:35), a conserved pattern of short introns in S.
cerevisiae (Davis et al., 200; Rymond & Rosbash, 1992).
Although introns are common in S. cerevisiae, they are rarely
present in Pichia pastoris (Cregg, personal communication). The
coding sequence of Pichia pastoris EF-2 is present in FIG. 3(a) (b)
(c) (d). It contains 2526 nucleotides and code for 842 amino acids.
The Pichia pastoris EF-2 is the same as the EF-2 of S. cerevisiae
and Schizosaccharomyces pombe in length and shares 88% of identity
in amino acid sequence with S. cerevisiae (Perentesis et al., 1992)
and 78% with S. pombe (Mita et al., 1997). Both S. cerevisiae and
S. pombe have two functional EF-2 genes (EFT1 and EFT2) per haploid
genome. These two copies of the EF-2 genes encode the same amino
acid sequence, but have a few different nucleotides (4 in S.
cerevisiae and 13 in S. pombe) in their coding regions and
dissimilar flanking sequences. However, the sequencing data of
independent clones derived from mRNA and genomic DNA showed that
all of the different clones had the same 5' and 3'end flanking
sequences and an identical coding sequence. This plus the evidence
of Southern blotting of restriction enzyme digested genomic DNA
shows that Pichia pastoris has only one copy of the EF-2 gene.
Example 4
Construction of Mutating Plasmid pBLURA-.DELTA.5'mutEF-2
To create DT resistant strains of Pichia pastoris, the EF-2 gene
was mutated so that the Gly at position 711 was changed to an Arg.
The strategy employed to introduce the mutation into the genome is
based on that described by Shortle et al. (1984) and is shown in
FIG. 4. In this method, a truncated form (at only one end) of the
targeted gene was used to introduce a mutation to the gene in the
genome by homologous recombination. Integration of the truncated
gene fragment bearing a mutation will lead to a situation that the
genome contains one intact copy of the gene with the mutation and
one truncated copy. Because the targeted site is located close to
the 3' end, the 5' truncated EF-2 (.DELTA.5'EF-2) was used as the
mutating sequence. .DELTA.5'EF-2 contained 1127 nucleotides from
the 3'end of EF-2 starting from position 1432 to 2549 (FIG. 3) and
was generated by PCR with Pfu polymerase. After cloning into the
pCR2.1-TOPO vector, .DELTA.5'EF-2 was mutagenized in vitro by
oligonucleotide-directed mutagenesis. The mutagenized .DELTA.5'EF-2
(.DELTA.5'mutEF-2) was then released from pCR2.1-TOPO by
restriction enzymes Nhe1 and Not 1 that cut at the 5' and 3' ends
respectively, and then cloned into the vector pBLURA-SX (provided
by Professor Cregg and described in Geoffrey et al. (2001)) that
had been digested by Nhe1 and Not 1. The vector contains the
auxotrophic marker URA3. Plasmid DNA pBLURA-.DELTA.5'mutEF-2
purified from bacterial was linearized before being electroporated
into the strains of Pichia pastoris. The plasmid DNA contains a
unique Aat II site located in the EF-2 sequence, about 220
nucleotides before the mutation site. Cleavage at this site will
target the plasmid integration to the EF-2 locus and favors the
event of the mutagenized sequence being transferred to the intact
copy of EF-2. Three uracil auxotrophic strains of Pichia pastoris
were transformed with the plasmid DNA. They are JC308 (ade1 arg4
his4 ura3), JC303 (arg4 his4 ura3) and JC307 (his4 ura3), and were
all provided by Professor Cregg and described in Geoffrey et al.
(2001). JC308 was transformed first followed by JO303 and
JC307.
Example 5
Identification of Clones Containing Mutated EF-2
After electroporation with the linearized pBLURA-.DELTA.5'mutEF-2
DNA, Cells were spread onto plates containing synthetic complete
medium for yeast minus uracil (K.D Medical, Maryland). Ura+ clones
were then analyzed by "Colony PCR" for the presence the correct
mutations in the intact copy of EF-2. In this method, yeast cells
from colonies were picked by tooth pickers and resuspended in 20 ul
of PCR mix. DNA released from the cells lysed by the first PCR step
(94.degree. C. for 5 minutes) served as the template for PCR
amplification. Five primers were used in the PCR detection
procedures: primers 5'EF-2 and 3'EF-2C were described previously in
section 4; EF-2 (1318) has the EF-2 sequence from position 1318 to
1341; primer wEF-2 is complementary to the positions 2100 to 2119,
whereas primer mEF-2 has the sequence complementary the same
positions but specific to the mutations The designed nucleotide
mutations shown in FIG. 1 created a new Sac II restriction enzyme
site that was used to confirm the correct mutations in the
genome.
Primers 5'EF-2 and mEF-2 were first used to detect the mutations in
the Ura+ transformants. FIG. 5a shows that 9 (clones 1, 2, 3, 12,
13, 14, 33, 40 and 41) of the 12 selected Ura+ clones of JC308 are
mEF-2 primer positive, they had a PCR product of the expected size
(about 2.2 kbp), whereas clones 25, 38 and 47 were negative. As
shown in FIG. 5b, when the same clones were analyzed for the
presence of wild type sequence with primers EF-2 (1318) and wEF-2,
all three mEF-2- clones were wEF-2 primer positive. A 0.8 kbp PCR
fragment was produced. All mEF-2+ clones were wEF-2- except for
clones 33 and 41 that were also wEF-2+. Finally when primers EF-2
(1318) and 3'EF-2C were used, all of the selected clones yielded a
PCR product of about 1.2 kbp as expected (FIG. 5c). The PCR
products from the clones that were mEF-2+ and wEF-2- were
completely digested by Sac II, whereas those of the clones 25, 38
and 47 that were mEF-2- and wEF-2+ were not cut by the enzyme. In
agreement with being both mEF-2+ and wEF-2+, clones 33 and 41
produced both Sac II clearable and non-clearable PCR produces. To
investigate why clones 33 and 41 had both mutated and wild type
EF-2, these clones were streaked on new selection plates and let
the cells grow to form colonies. Ten well-isolated colonies were
picked from each and performed the PCR with primer EF-2 (1318) plus
primer 3'EF-2C and the Sac II digestion steps. None of the colonies
had the same mixed PCR products as the originals. PCR products of 4
colonies from clone 33, 7 from 41 were completely digested by Sac
II, whereas those of other colonies from clones 33 and 41 were not
cut at all. This experiment shows that clones 33 and 44 were each
originally formed by two different cells, one had an intact EF-2
with the mutations, and the other had an intact wild type EF-2.
This experiment was then repeated and checked some of the clones
that had only the Sac II clearable EF-2 (clones 1, 2, 3, 12, 13,
14, and 40) and confirmed that they only contained the mutated
intact EF-2. After the success in obtaining EF-2 mutant clones of
JC308, the same selection procedure was used to identify EF-2
mutant clones of JC303 and JC307. Among the Ura+ positive clones
picked for analysis, 35% of them contained only the mutated intact
EF-2. This high frequency of complete mutation may be due to the
fact that Pichia pastoris only has one copy of EF-2 per haploid
genome. As shown for CHO cells and S. cerevisiae, the Arg
substitution for Gly711 of EF-2 in Pichia pastoris did not affect
cell growth at normal conditions.
Example 6
Expression of DT A Chain in the EF-2 Mutants
To test whether the obtained EF-2 mutants are resistant to DT
expression, mutEF2JC307-8, an EF-2 mutant clone (clone 8) of JC307,
was transformed with the plasmid DNA of pPIC3-DtA. The construction
of pPIC3-DtA was previously described (Woo et al., 2002). Briefly,
the DT A chain gene with BamH I at its 5' end and Not I at 3' was
amplified by PCR, inserted into Pichia pastoris expression vector
pPIC 3 (Invitrogen) and digested with these two enzymes.
Integration of pPIC3-DtA allows cytosolic expression of DT chain
upon methanol induction. This plasmid DNA had previously been used
to transform the GS200 strain of Pichia pastoris (Invitrogen) and
two of the resulting clones (C3 and C4) were used in the study on
tolerance of Pichia pastoris to DT (Woo at al., 2002). C3 had been
characterized as a non-DT A expressing clone, whereas C4 is a DT A
expressing clone. After the transformation with pPIC3-DtA, six
mutEF2JC307-8, (mutEF2JC307-8-DtA(1) to (6), clones were randomly
picked for analysis of their cytosolic expression of DT A chain and
their viability after methanol induction. Cells from single
colonies of mutEF2JC307-8-DtA(1) to (6), C3 and C4 were grown in 2
ml YPD (Yeast extracts-Peptone-Dextrose) medium at 30.degree. C.
overnight before being pelleted down by centrifugation. Cells from
each culture were resuspended in YP medium to a density at OD600
nm.+-.0.5. Cell suspensions (2 ml) were induced by adding methanol
to 1% and incubated at 30.degree. C. with vigorous shaking. After
methanol induction for 24 hours, cells from 100 .mu.l of each
culture were pelleted down and washed with PBS buffer. After this,
cells were resuspended in PBS and mixed with protein sample buffer.
Finally, the samples were subjected to two cycles of boiling and
freezing on dry ice before being analyzed by SDS-PAGE and Western
blotting with a DT specific antibody. The cultures of
mutEF2JC307-8-DtA(3) and (5), C3 and C4 were also used for
viability assay. This was performed by diluting each culture 104 to
107 fold with PBS buffer, plating 100 .mu.l of aliquot on YPD plate
and then counting the colonies appearing on the plates after 3 days
incubation at 30.degree. C. The result of SDS-PAGE and Western
blotting showed that except for mutEF2JC307-8-DtA(5), all
mutEF2JC307-8-DtA clones expressed DT A chain (FIG. 6a). The
expression of mutEF2JC307-8-DtA(3) was estimated roughly at 20
.mu.g/ml cell culture. As expected, C3 did not express DT A.
Although C4 did express DT A, the protein band was barely visible
(FIG. 6b). Before methanol induction, the number of the colony
forming units (CFU) per ml of cells was about the same for
mutEF2JC307-8-DtA(3) and (5), C3 and C4. After 24 hours methanol
induction, the CFU number of mutEF2JC307-8-DtA(3) and (5) and C3
all increased about 103 fold, whereas the CFU number of C4
decreased about 102 (FIG. 7). This result demonstrated that the
expression of DT A chain in the cytosol of cells bearing the
mutated EF-2 was not toxic to the cells.
Example 7
Small-Scale Expression in Shake-Flask Culture
MutEF2JC307-8 was first used to express the bivalent immunotoxin.
Since this EF-2 mutant is auxotrophic for histidine, it was
transformed with plasmid pPIC9K containing the final version of the
modified gene for the bivalent immunotoxin: A-dmDT390-bisFv
described in Woo et al. (2002). Bivalent refers to two repeats of
the sFv antibody fragment. The protocols used for transformation,
selection for transformants, and protein expression and analysis
were described previously (Woo et al., 2002, which is incorporated
herein by reference in its entirety for the methods taught
therein). After transformation, 12 colonies were randomly picked
and analyzed for protein expression. SDS-PAGE analysis revealed
that all of the selected clones expressed the bivalent immunotoxin,
although some clones, such as clone number 2 [mutEF2JC307-8(2)],
expressed at slightly higher levels than others. When they were
cultured and expressed under the same conditions and at the same
time, mutEF2JC307-8 (2) expressed the bivalent immunotoxin at the
same level as pJHW#2, a clone of GS 115 (bearing the wild type
EF-2) that had been transformed with pPIC9K-A-dmDT390-bisFv. The
expression levels of mutEF2JC307-8(2) and pJHW#2 were about 5 to 10
.mu.g/ml of culture supernatant in shake-tube culture. The fact
that mutEF2JC307-8(2) did not yield a higher level of expression
demonstrated that other factors in addition to EF-2 ADP
ribosylation also limit production of the bivalent immunotoxin.
In a second attempt to express the bivalent immunotoxin in mutated
Pichia pastoris, two copies of A-dmDT390-bisFv gene were introduced
into mutEF2JC303-5, an EF-2 mutant clone (clone 5) of JC303, which
is auxotrophic for histidine and arginine. To build an expression
vector with ARG4 selection marker, The A-dmDT390-bisFv gene (see
FIG. 20) was cloned into the expression vector pBLARG-SX3 provided
by Professor Cregg and described in Geoffrey et al. (2001). This
was done by inserting the final version of A-dmDT390-bisFv gene
plus the .alpha.-factor signal sequence released from pPICZ.alpha.
(Woo et al., 2002) by Hind III and Not I digestion into pBLARG-SX3
that had been cut with these two restriction enzymes. The resulting
construct, pBLARG-A-dmDT390-bisFv (FIG. 8a), together with
pPIC9K-A-dmDT390-bisFv were electroporated at the same time into
mutEF2JC303(5). Transformants expressing these two marker genes
were selected on plates containing synthetic complete medium minus
arginine and histidine (K.D Medical, Maryland). Eighteen colonies
were picked from the selection plate and analyzed for their
expression of the immunotoxin protein. SDS-PAGE showed that they
all secreted roughly the same amount of intact immunotoxin protein
into induction media. This amount was similar to that secreted from
single copy clones: mutEF2JC307-8(2) and JHW#2. As shown in FIG.
9a, three of the selected clones (clones 3, 6, 8) also expressed a
smaller, but much more abundant protein that reacted with an
anti-DT antibody and had the same size as the monovalent
immunotoxin (Liu et al., 2000). The smaller protein is more stable
than the intact protein regardless as to whether this protein was
produced from a truncated copy of A-dmDT390-bisFv gene or the
proteolytically cleaved product of the intact protein. The figure
also shows that there were many other smaller proteins in the
culture supernatant that reacted with the anti-DT antibody; they
were most likely the proteolytic cleaved products of the intact
protein. The smallest and also the most abundant one was
characterized as the A chain of DT, which is very stable (Collier
1975) and can account for the final product of proteolytic
degradation of the intact protein. The degradation also took place
inside the cell (FIG. 9b). Because the A chain is about 1/4 of the
size of the intact protein, the amount of the A chain shown on the
Western blot indicates that the actual expression level was
probably several times higher than the level of intact protein
present in the induction medium. A majority of the protein
synthesized was probably degraded either before or after secretion
out into the medium. Although the double copy clones accumulated
the same amount of intact protein in the medium as the single copy
clones, the double copy clones produced a larger amount of degraded
products, indicating that more gene products had been synthesized.
Different measures to control the protein degradation have been
employed but the production of the intact protein has not been
increased. Thus protein degradation either within or outside the
cell is a limiting factor to increase the production of the
bivalent immunotoxin.
Example 8
Alternative Method for Large-Scale Expression in Fermentation
Culture Using PMSF
For large scale cultures, the BioFlo 4500 fermentor (New Brunswick
Scientific Company), which was installed with a methanol sensor
(Raven Biotechnology Company) for precise control of methanol
concentration in cultures, was used. The initial fermentation
medium (10 L) contained 1% yeast extract, 2% peptone or 2% soytone,
4% glycerol, 1% casamino acids, 1.34% yeast nitrogen base with
ammonium sulfate and without amino acids, 0.43% PTM1 salt solution
and 0.01% antifoam 289 (Sigma Co.) or a mixture of antifoam 204,
0.01% and Stuktol 0.01%. Depending on culture conditions, 75% (v/v)
glycerol solution having 1.8% PTM1 salt solution was used for
obtaining a desired cell density before methanol induction and/or
supplementing an additional carbon source or energy source for
methanol induction. 100% methanol solution for induction containing
20 mM PMSF and/or 1.2% PTM1 salt solution was used. Alternatively,
induction was performed with a continuous feed of 4:1
methanol/glycerol containing 73 mM PMSF, and PMSF was added to 1 mM
final concentration just prior to induction. In order to prepare a
seed culture for the fermentor, 50 ml of YPD (1% yeast extract, 2%
peptone and 2% glucose) was innoculated with 1 ml of a frozen stock
of YYL #8-2 and then cultivated for 2 days at 30.degree. C. with
vigorous shaking. The 30 ml from the 50 ml culture was used as the
first seed culture for inoculating approximately 600 ml of the
second seed culture. The DO level in the fermentor was maintained
at more than 25% for the whole fermentation run. The pH in the
fermentor was kept at 3.5 for growth phase and 7.0 for methanol
induction phase. The temperature was set at 28.degree. C. for
growth and 15-25.degree. C. for methanol induction. Casamino acids
solution (20%) was fed continuously at 20 ml/h during methanol
induction or at the maximum speed of a pump for feeding for the
first 2 hours of methanol induction. At the temperature of
23.degree. C. for methanol induction, the expression level of the
bivalent immunotoxin was the highest among 4 different runs.
However, its expression level was similar to that of the current
expression strain, pJHW #2. Table 1 summarizes results of 5
fermentation runs.
TABLE-US-00006 TABLE 1 Results of Fermentation Runs Run 1 Run 2 Run
3 Run 4 Run 5 (#27) (#28) (#29) (#36) (#41) glycerol-fed 5.5 4 0
7.5 6 batch time (hour) cell density 19.44 18.60 11.93 20.02 21.16
at the start of methanol induction (%) final conc. 2.sup.1 2.sup.1
2.sup.1 7.sup.2 2.sup.1 of PMSF (mM) casamino 100.sup.3 100.sup.3
100.sup.3 100.sup.4 138.sup.5 acid (g) temperature 25 20 15 23 23
for methanol induction (C.) methanol 3093 2776 2474 2538 3000
consumption (g) glycerol 475 0 0 0 0 feeding for methanol induction
(g) methanol 43 44 70 44 94 induction time (hour) final volume 13.3
12.3 11.9 11.4 13.4 of the supernatant (L) expression 10 15 10 15
NM.sup.6 level (mg/L) at 22 hours of induction expression NM NM NM
NM 27.5 level (mg/L) at 42 hours of induction expression NM NM NM
NM 30.0 level (mg/L) at 66 hours of induction Expression 3.3 18.3
26.6 27.5 32.5 level (mg/L) at harvest Total amount 43.9 225.1
316.5 313.5 435.5 of the bivalent immunotoxin (mg) .sup.150 ml of
PMSF solution (3.484 g per 50 ml of methanol) was fed on the based
of methanol concentration in the culture for the beginning of
methanol induction. After the finish of feeding of PMSF solution,
methanol solution containing 12 ml of PTM1 salt solution per 1
liter of methanol was replaced. .sup.215 ml of PMSF solution (1.742
g per 15 ml of methanol) was injected at the beginning of methanol
induction. On the basis of methanol concentration, methanol
solution (20 mM PMSF and 12 ml of PTM1 salt solution/liter of
methanol) was fed. .sup.310% casamino acids solution was fed at the
maximum speed of a pump at the start of methanol induction.
.sup.420% casamino acids solution was continuously fed at 20
ml/hour of pump speed. .sup.515% casamino acids solution was
continuously fed at 20 ml/hour of pump speed. .sup.6not
measured
Under these conditions, maximum production of the wild-type
expression strain, pJHW #2, is 27.5 mg/L with the total amount of
286.0 mg of the bivalent immunotoxin in 42 hrs of methanol
induction. This level could not be increased beyond 42 hrs of
induction. However, under conditions adopted from those for pJHW
#2, production the EF-2 mutant strain YYL8-2 continued to increase
up to 94 hrs after methanol induction in spite the fact that the
initial 10 L of culture medium was gradually diluted to 13.4 L with
methanol and 10% casamino acids solution (see run 5). The total
amount of the bivalent immunotoxin of run 5 was 435.5 mg (32 mg/L).
This is 1.46-fold greater that the maximum production of pJHW #2.
The difference in the production of the bivalent immunotoxin
between these two strains is reflected by the methanol consumption
rates as shown in FIG. 10.
Example 9
Previous Method of Purification of the Bivalent Immunotoxin
The Pichia pastoris supernatant contains materials that compete
with A-dmDT390-bisFv in binding to anion exchange resins. In
addition, the toxin moiety can not be exposed to pH less than 6.5
without undergoing unfolding of hydrophobic residues. Therefore a
hydrophobic interaction chromatographic step using Butyl-650M
(TosoHaas) was employed. This resin preferentially binds monomeric
A-dmDT390-bisFv over the dimeric form, a species having greatly
diminished biologic activity. The capture step also concentrates a
Pichia pastoris glycoprotein that appears as a diffuse band of
.about.40 kD on SDS gels but has the same mobility as
A-dmDT390-bisFv under size exclusion chromatography. This material
is eliminated by preferentially binding to Con A Sepharose
(Pharmacia). A Superdex (Pharmacia) size exclusion step eliminates
any A-dmDT390-bisFv dimmer not previously screened during the
capture step. The overall yield is 45% when the fermentation
conditions achieve an A-dmDT390-bisFv monomer content of 85%. The
procedure for purification of A-dmDT390-bisFv is presented below:
1. Butyl-650M hydrophobic interaction chromatography Bed volume:
600 ml (in 10 cm diameter column) Flow rate: 50-70 cm/hour sample
preparation: solid sodium sulfate and 1 M Tris buffer (pH 8.0) were
added to the final concentration of 0.5 M and 20 mM, respectively.
sample volume: typically 10 L binding buffer: 500 mM Na2SO4, 1 mM
EDTA, 20 mM Tris buffer (pH 8.0) elution buffer: 5% glycerol, 1 mM
EDTA, 20 mM Tris buffer (pH 8.0) procedure: equilibrate the column
with binding buffer applied the sample onto the column washed with
5 bed volume of binding buffer eluted A-dmDT390-BisFv with 6 bed
volume of elution buffer regenerated the column by manufacturer's
protocol volume of eluted fractions: 3600 ml 2. Diafiltration
membrane: Amicon spiral-wound membrane (30 Kd) model S3Y30 (0.23
m2) sample: eluted fractions from capturing step diafiltration
buffer: 5% glycerol, 1 mM EDTA, 20 mM Tris buffer (pH 8.0) buffer
volume used for diafiltration: 6 volume of the sample pressure: 7
psi final volume: around 2 L 3. Poros 50 HQ ion exchange
chromatography Bed volume: 40 ml (in 2.6 cm diameter column) Flow
rate: 1 ml/min sample: diafiltrated sample (typically 2 L) binding
buffer: 5% glycerol, 20 mM Tris buffer (pH 8.0) elution:
0.about.500 mM NaCl gradient (10 bed volume) in binding buffer
procedure: equilibrate the column with binding buffer applied the
sample onto the column washed with 3 bed volume of binding buffer
and started to collect 20 ml of each fraction eluted
A-dmDT390-BisFv with 10 bed volume of 0.about.500 mM NaCl gradient
regenerated the column by manufacturer's protocol fraction size: 20
ml 4. Con A affinity chromatography sample: 90.about.120 ml of the
eluted fractions having A-dmDT390-BisFv from Poros IEX bed volume:
60 ml resin packed in 2.5 cm.times.20 cm column binding buffer: 5%
glycerol, 20 mM Tris buffer (pH 8.0) flow rate: by gravity
procedure equilibrated the column with binding buffer applied the
sample to the column and started to collect 10 ml of each fraction
added 0.5 M EDTA to each fraction at the final conc. of 1 mM washed
the column with 5 bed volume of binding buffer regenerated the
resin by manufacturer's protocol 5. Superdex 200 prep grade Gel
filtration sample: 50 ml pooled fraction containing A-dmDT390-BisFv
from Con A affinity step sample preparation: 5 M NaCl was added to
the final conc. of 200 mM bed volume: 970 ml of Superdex 200 resin
in 5 cm.times.60 cm column buffer: 200 mM NaCl, 1 mM EDTA, 20 mM
Tris-Cl (pH 8.0) and 5% glycerol flow rate: 1 ml/min procedure
equilibrated the column with binding buffer applied the sample to
the column and started to collect 20 ml of each fraction eluted the
column with 1 bed volume of the buffer regenerated the resin by
manufacturer's protocol
This method is difficult from a regulatory standpoint because Con
A, which is toxic is leached from the column matrix. In contrast,
the present method (see Example 16 and Example 38) uses borate to
eliminate the glycoprotein. Borate binds to the glycoprotein vicyl
hydroxyl groups and imparts a negative charge thus making the
glycoprotein stick tighter to the anion exchange column. However
the rIT as no carbohydrate groups and is eluted by the borate.
Example 10
Construction of Expression Vectors pPGAP-Arg and pPGAP-His
The promoter for Pichia pastoris glyceraldehydes-3-phosphate
dehydrogenase gene (P.sub.GAP) has been characterized and used for
heterologous protein expression in Pichia pastoris (Waterham et
al., 1997). P.sub.GAP is a strong and constitutive promoter. It was
reported that protein expression under control of P.sub.GAP in
glucose-grown Pichia pastoris was higher than that of the commonly
used P.sub.AOX1 in methanol-grown cells (Waterham et al., 1997;
Doring et al., 1998). The disadvantage of constitutive promoters in
heterologous protein expression is that they are not suitable for
proteins that are toxic to the expressing host. Since the EF-2
mutants of Pichia pastoris were resistant to cytosolic expression
of DT A, these mutants should allow constitutive expression of DT
or PE based immunotoxins in their cells. Therefore P.sub.GAP was
used to drive the expression of A-dmDT390-bisFv in Pichia pastoris
in the hope that the P.sub.GAP would increase the expression level
of protein.
The construct pPGAPArg-A-dmDT390-bisFv was made by replacing the
AOX1 promoter of pBLARG-A-dmDT390-bisFv with P.sub.GAP (FIG. 8b).
First, P.sub.GAP was amplified from the expression vector pGAPZ A
(Invitrogen) by PCR with primers containing sequences of P.sub.GAP
5' and 3' ends. The 5' and 3'end primers had a Nhe I and Hind III
added respectively. After digestion with Nhe I and Hind III, the
PCR products of P.sub.GAP were then inserted in
pBLARG-A-dmDT390-bisFv that had been cut with these two restriction
enzymes to remove the AOX1 promoter. The construct
pPGAPHis-A-dmDT390-bisFv (FIG. 8c) was created by joining DNA
fragments from plasmids pPIC9K (Invitrogen) and
pPGAPArg-A-dmT390-bisFv. The plasmid pPIC9K was first cut by Sfu I,
after filling in with Klenow Fragment by Not I, then the DNA
fragments were separated by agarose gel electrophoresis. The 5.1
kbp fragment containing kanamycin resistant gene, HIS4 gene and 3'
AOX1 transcription termination (TT) was isolated and ligated with
the plasmid DNA pPGAPArg-A-dmDT390-bisFv that had been digested
with Not 1 and Sca I to remove the 3'AOX1 TT and ARG4 gene.
Example 11
Expression of the Bivalent Immunotoxin Under the Control of
P.sub.GAP
As done for the expression under AOX1 promoter, one copy clones
were obtained by transforming mutEF2JC307-8 with construct
pPGAPHis-A-dmDT390-bisFv; two copy clones by transforming
mutEF2JC303-5 with both pPGAPArg-A-dmDT390-bisFv and
pPGAPHis-A-dmDT390-bisFv. This time the two copy clones were
constructed by two steps. First, mutEF2JC303-5 was transformed with
pPGAPArg-A-dmDT390-bisFv, after selection and protein expression
analysis. The clone that produced the intact immunotoxin at highest
level was then transformed with pPGAPHis-A-dmDT390-bisFv.
Small scale protein expression was carried out by inoculating a
single colony to 2 ml YPD, and after overnight growth, cells were
seeded in 2 ml expression medium at an OD.sub.600 nm=0.5, and then
incubated at 28.degree. C. for 24 hours before the culture
supernatant was analyzed for expression of the immunotoxin. The
expression medium is the similar to BMMYC used for expression of
the immunotoxin under P.sub.AOX1, but instead of 0.5% methanol it
contains 2% glucose. SDS-PAGE analysis showed that accumulation of
the intact protein in the culture supernatant of 2 copy clones was
slightly higher than that of 1 copy clones. One of the 2 copy
clones (Pgap2-9) has consistently producing 10 to 15 .mu.g of
intact protein per ml of culture medium. The results of Western
blotting analysis of culture supernatant and extract cell pellet
were consistent with those obtain from the expression under
P.sub.AOX1.
The production of the bivalent immunotoxin under control of
P.sub.GAP was slightly higher than that under P.sub.AOX1 in shake
tube culture. Since fermentation allowed cells to grow to very high
density, the increase in production under control of P.sub.GAP may
be more significant when the production is in a bioreactor. The
other advantage of P.sub.GAP controlled expression is that
production procedure was simpler and shorter. It did not require
addition and maintenance of methanol in the expression medium. The
whole production procedure was about 40 hours compared to more than
72 hour for that of the P.sub.AOX1 controlled expression.
Example 12
Yeast Strains and Strain Maintenance
In order to optimize fermentation conditions, genetically
engineered Pichia pastoris strain JW102 (former name was pJHW #2)
was used, which was generated for production of the bivalent
immunotoxin from the host strain GS115 (Invitrogen, Carlsbad,
Calif.) (Woo et al., 2002). The AOX1 (alcohol oxidase 1) promoter
controlled the expression of immunotoxin during methanol induction.
The gene product was secreted by the alpha-prepro leader sequence.
To compare the growth profile and fermentation parameters in the
fermentor, X-33 and JW103 (MutS) or mutEF2JC307-8(2) were used
(Table 2) and elsewhere.
TABLE-US-00007 TABLE 2 The Pichia pastoris strains used in this
study. Names Protein of interest Phenotypes JW102* Secretion of
bivalent immunotoxin His.sup.+Mut.sup.+ JW103* Secretion of
bivalent immunotoxin His.sup.+Mut.sup.S C-4 Cytosolic expression of
A chain of DT His.sup.+Mut.sup.+ X-33 Host strain
His.sup.+Mut.sup.+ *JW102 and JW103 were renamed from pJHW#2 and
pJHW#3, respectively (Woo et al., 2002)
Strain JW102, expressing the bivalent immunotoxin, was genetically
very stable. After subculturing the strain more than 60 times onto
YPD plates (1% yeast extract, 2% Bacto peptone, 2% dextrose and 2%
agar), the strain maintained expression of the bivalent
immunotoxin. A colony isolated at the very early stage was expanded
in YPD broth (1% yeast extract, 2% Bacto peptone, 2% dextrose) and
then kept as frozen stock at -80.degree. C. Frozen stock was
prepared by mixing a 2-day incubation culture with an equal volume
of 25% (v/v) glycerol and 1 ml of the mixture was dispensed into a
2 ml Cryo vial.
Example 13
Fermentation
A BioFlo 4500 fermentor (New Brunswick Scientific Company, Edison,
N.J.), with a methanol sensor and controller (Raven Biotechnology
Company, Canada) that maintained methanol at 0.15% (v/v) during
induction was used. This fermentor was linked to a computer running
an AFS-BioCommand Windows-based software (New Brunswick Scientific
Company), which allowed for the control of all parameters by
programmed processes. The basic initial fermentation medium (10
liters) contained 2% (20 g/L) yeast extract, 2% (20 g/L) Soytone
Peptone (Difco), 4% (40 g/L) glycerol, 1.34% (13.4 g/L) yeast
nitrogen base with ammonium sulfate and without amino acids, 0.43%
(4.3 ml/L) PTM1 salt solution and 0.02% (v/v) antifoam 289 (Sigma
Co.). The PTM1 salt solution (Invitrogen) contained of 24.0 mM (6
g/L) cupric sulfate (CuSO.sub.4.5H.sub.2O), 0.534 mM (80 mg/L)
sodium iodide (NaI), 17.8 mM (338.6 mg/L) manganese sulfate
(MnSO.sub.4.5H.sub.2O), 0.827 mM (200 mg/L) sodium molybdate
(NaMoO.sub.4.2H.sub.2O), 0.323 mM (20 mg/L) boric acid
(H.sub.3BO.sub.3), 2.1 mM (500 mg/L) cobalt chloride
(CoCl.sub.2.6H.sub.2O), 147.0 mM (20 g/L) zinc chloride
(ZnCl.sub.2), 234.0 mM (65.1 g/L) ferrous sulfate
(FeSO.sub.4.7H.sub.2O), 1.64 mM (400 mg/L) biotin, 188.0 mM (18.4
g/L) sulfuric acid (H.sub.2SO.sub.4).
The glycerol batch phase was completed within 18 h of inoculation,
and complete consumption of glycerol in the culture was detected by
monitoring the DO spike. A glycerol-fed batch phase ensued, during
which 75% (v/v) glycerol was fed by ramping up the feeding rate at
0.1 g/min to 3.0 g/min for 7 h. Seventy-five percent (v/v) glycerol
solution containing 18 ml/L (1.8%) of PTM1 salt solution was used
for obtain the desired cell density for 7 h before methanol
induction. Induction was performed with a continuous feed of
methanol or 4:1 methanol:glycerol (based on volume) with or without
10 mM PMSF (phenylmethylsulfonyl fluoride). The feeding rate of
methanol or 4:1 methanol:glycerol was automatically controlled to
be maintained at the set point (0.15% (v/v) methanol in the
culture) by the methanol sensor and controller. The methanol
consumption rate was measured by weighing a methanol solution or
methanol/glycerol mixed solution every one minute on a computer
interfaced balance (PG5002S, Mettler Toledo, Switzerland). PMSF was
added to 1 mM final concentration just prior to induction when PMSF
was added during methanol induction. A casamino acids or yeast
extract solution (10%, w/v) was fed continuously at 10 ml/h/10 L
initial volume during methanol induction.
Alternatively, with the EF-2 mutant, the carbon source may be
limited to methanol during induction and the methanol feed rate may
be limited to about 0.5-0.75 ml/min or lower and regulated by a
precision pump (Table 3). In run #53, methanol was fully fed by a
pump that was controlled by a methanol sensor to maintain a set
point of 0.15% methanol in the culture. In run #56, methanol
feeding during methanol induction was limited to 0.75 ml/min.
Concentration of bivalent immunotoxin in the supernatants taken at
various induction time points was determined on Coomassie-stained
SDS-polyacrylamide gels. For further comparison between both runs,
protein yield of the Butyl 650M HIC capture step was determined
from 1 liter of each supernatant. Limited feeding of methanol
during methanol induction increased the secretion level of bivalent
immunotoxin up to 50 mg/L.
Table 3. Limited feeding of methanol at a rate of 0.75 ml/min
during methanol induction increased secretion level of bivalent
immunotoxin in the EF-2 mutant strain.
TABLE-US-00008 Run #53 Run #56 Induction Full feeding Limited
feeding time Purification step of methanol of methan 22 hr
Supernatant 12.5 mg/L 15.0 mg/L Butyl 650M HIC 11.7 mg 14.4 mg
(from 1 L supernatant) 44 hr Supernatant 30.0 mg/L 35.0 mg/L Butyl
650M HIC 23.4 mg 28.8 mg (from 1 L supernatant) 67 hr Supernatant
35.0 mg/L 50.0 mg/L Butyl 650M HIC 29.3 mg 40.3 mg (from 1 L
supernatant)
In order to prepare a seed culture for the fermentor, 50 ml of YSG
broth (1% (w/v) yeast extract, 2% (w/v) Soytone Peptone, 1% (w/v)
glycerol) was inoculated with 1 ml of a frozen stock (-80.degree.
C. in 25% (v/v) glycerol) and then cultivated for 2 days at
28.degree. C. at 250 RPM (orbit diameter, 1.9 cm).). Thirty ml from
a 50 ml culture was used as the first seed culture for inoculating
600 ml of YSG broth in two 1 L flasks. After cultivation for 1 day
at 28.degree. C. at 250 rpm (orbit diameter, 1.9 cm), the cultures
were used as the second seed culture for inoculation of 10 L of
initial complex fermentation medium in the fermentor. All
parameters were automatically managed by running processes
programmed in the AFS-BioCommand software. The DO level in the
fermentor was maintained at >40% for the entire fermentation
with O.sub.2 supplementation as needed. The pH in the fermentor was
kept at 3.5 during the growth phase and at 7.0 during the methanol
induction phase by adding 29% (v/v) NH.sub.4OH or 40% (v/v)
H.sub.3PO.sub.4. The pH was ramped up from 3.5 to 7.0 for 2 h
before the initiation of methanol induction. The pH shifting
procedure reduced the secretion of contaminant proteins (75 kDa and
35 kDa bands) into the supernatant. The temperature was set at
28.degree. C. for growth and 15-25.degree. C. during methanol
induction. The induction temperature was ramped down from 28 to
25-15.degree. C. during the first 4 h of methanol induction.
Reducing the bioreactor agitation may increase the fraction of
monomeric and bioactive immunotoxin. A bioreactor agitation of 400
rpm increases the fraction of monomeric and bioactive immunotoxin
by 50% over a bioreactor agitation of 800 rpm (FIG. 12). Providing
a detergent or other denaturant during agitation may reduce
aggregation of the immunotoxin. Including TWEEN 20.RTM. at 0.01%
during agitation of immunotoxin further reduces aggregation and
increases the fraction of monomer and bioactive immunotoxin to 90%
(FIG. 13). After harvesting the culture, the supernatant was
prepared by centrifugation (2,800.times.g at 4.degree. C. for 30
min). EDTA was added to a final concentration of 5 mM to prevent
protein degradation during storage at 4.degree. C.
Example 14
Measurement of Wet Cell Density (%, w/v) for Monitoring Cell
Growth
One ml of culture sample was placed in a tared 1.5-ml
microcentrifuge tube and spun at 20,800.times.g at 25.degree. C.
for 2 min. The supernatant was removed with a pipet and residual
liquid in the tube was blotted with filter paper. After weighing
the tube containing the cell pellet, the wet cell density (%, w/v)
was calculated.
Example 15
Purification
A scaleable 3-step procedure for purification of the bivalent
immunotoxin has been developed that utilizes borate anion exchange
chromatography to eliminate contaminating host glycoproteins.
Purifications were performed with 1 L of centrifuged supernatant.
No dialysis or diafiltration step was employed. In brief, 1 L of
supernatant was mixed with 28.4 g of solid Na.sub.2SO.sub.4 and
applied to a 100 ml bed of butyl-650M and eluted with 5% glycerol,
20 mM tris and 1 mM EDTA, pH 8.0, after washing in 200 mM
Na.sub.2SO.sub.4. 600 ml of eluant was diluted with 4.2 L of TE
buffer (20 mM tris, 1 mM EDTA, pH 8.0) and applied to a 40 ml bed
of Poros 50 HQ. The bivalent immunotoxin was eluted in steps of
sodium borate buffer from 25-100 mM, and then glycoproteins and
some highly aggregated immunotoxin were eluted with 1 M NaCl. 1.2 L
of the borate eluant was diluted with 3.6 L of TE buffer and
applied to a 5 ml prepacked bed of Hi-trap Q. After washing, the
bivalent immunotoxin was eluted with a 0-400 mM NaCl gradient.
Butyl-650M hydrophobic interaction chromatography (HIC):
Approximately 100 ml of Butyl-650M resin (Tosoh Biosep LLC) was
packed in a 5 cm.times.20 cm XK column (Amersham Pharmacia Biotech)
and the column was equilibrated with Buffer A containing 200 mM
Na2SO4, 1 mM EDTA, 20 mM Tris-Cl buffer (pH 8.0). Solid sodium
sulfate and 1 M Tris-Cl buffer (pH 8.0) were added to 1 liter of
the supernatant to a final concentration of 200 mM and 20 mM,
respectively. The sample was filtered with a 802 fluted filter
paper (>15 urn particle retention: Whatman Inc.; Clifton, N.J.,
USA) before loading. The flow rate was 44 cm/hour (14.4 ml/min).
After equilibrating the column, 1 L of the prepared sample was
applied onto the column, and then the column was washed with 6
column volumes of binding buffer A. The bound proteins to
Butyl-650M resin were eluted with 6 column volumes of Buffer B
containing 5% glycerol, 1 mM EDTA, 20 mM Tris-Cl buffer (pH 8.0).
The eluted fractions having the immunotoxin were pooled for the
next step (volume: 600 ml). After each run, the column was
regenerated according to the manufacturer's protocol. All steps
were performed in a cold room except for the first step that was
carried out at room temperature.
Poros 50 HQ anion exchange chromatography (AEX) by step-elution
with sodium borate buffer: Approximately 40 ml of Poros 50 HQ resin
(PerSeptive Biosystems) was packed in a 2.6 cm.times.20 cm XK
column (Amersham Pharmacia Biotech) and then the column was
equilibrated with Buffer B. The pooled sample from the previous
step was diluted with 4.2 L of TE buffer (20 mM Tris-Cl, 1 mM EDTA,
pH 8.0). The diluted sample was loaded onto the column at a flow
rate of 80.2 cm/hour (7.08 ml/min), and then the column was washed
with 6 column volumes of Buffer B. The bound proteins were eluted
in steps of sodium borate of 25 mM, 50 mM, 75 mM and 100 mM in
Buffer B (10 column volumes for each step). These eluted fractions
were pooled for the next step. The residual protein bound to the
resin was stripped with 6 column volumes of 1 M NaCl in Buffer B.
After each run, the column was washed with 0.5 M NaOH and then
re-equilibrated with Buffer B minus 5% glycerol for the next
use.
Hi-trap Q anion exchange chromatography: A prepacked Hi-trap Q
anion exchange column (5 ml) was purchased from Amersham Pharmacia
Biotech. The pooled sample from the previous step was diluted with
3.6 L of TE buffer. The sample was loaded onto the equilibrated
column with Buffer B at a flow rate of 221.5 cm/hour (7.08 ml/min).
The column was washed with 5 column volumes of Buffer B. The bound
immunotoxin was eluted with a linear 0.about.400 mM NaCl gradient
in Buffer B (20 column volume). The flow rate for washing and
eluting steps was 2 ml/min and fraction size was 5 ml.
Example 16
Measurement of Protease Activity in the Supernatant
Unnicked CRM9 (one point mutation in the recognition domain of
diphtheria toxin (7)) was used as the substrate for measurement of
serine-protease activity in the supernatant at a final
concentration of 225 .mu.g/ml. The supernatant was incubated at
28.degree. C. with shaking at 250 rpm (orbit diameter, 1.9 cm) for
20 h before applying to a 4-20% tris-glycine precast SDS-PAGE gel
in the presence of reducing agent (100 mM dithiothreitol). CRM9
contains a well exposed furin/Kex-2 cleavage site between the A
fragment (22 kDa) and B fragment (40 kDa) spanned by a disulfide
bond. Protease activity in the medium was detected by loss of
unnicked CRM9 under reducing condition, and the band intensity of
the unnicked CRM9 was quantified by densitometry on Coomassie
stained gels.
Example 17
SDS-PAGE and Western Blotting
Proteins in culture supernatants were subjected to SDS-PAGE
utilizing tris-glycine 4-20% precast gels (Invitrogen) under
non-reducing and/or reducing conditions. For Western blotting, the
fractionated proteins were transferred onto nitrocellulose
membranes by electroblotting. Non-specific binding was blocked with
5% nonfat skimmed milk in TBST buffer (50 mM Tris-HCl, pH 7.5, 150
mM NaCl, and 0.1% TWEEN 20.RTM.). Goat polyclonal antibody directed
against diphtheria toxin (Thompson et al., 1995) diluted 1:2000 was
used as the primary antibody, and alkaline phosphatase-conjugated
rabbit anti-goat IgG (Roche Molecular Biochemicals) diluted 1:5000
was used as the secondary antibody. The immunotoxin was visualized
with one-step NBT/BCIP substrate (Pierce Chemical Company).
Alternatively, rabbit polyclonal antibody directed against
(G.sub.4S).sub.3 linker was used as the primary antibody for
detecting intact immunotoxin and degraded products since the
bivalent immunotoxin contained three (G.sub.4S).sub.3 linkers. This
antibody was raised against the synthetic peptide, whose amino acid
sequence was GGGGSGGGGSGGGGS (SEQ ID NO: 17).
Example 18
Cytotoxicity Assay
The tests to measure the specific cytotoxicity of anti-human
anti-CD3 immunotoxins expressed in Pichia pastoris were performed
as described (Neville et al., 1992). Briefly, immunotoxins were
applied to Jurkat cells, a human CD3.epsilon.+ T cell leukemia
line, (5.times.104 cells/well) in 96-well plates in leucine-free
RPMI 1640 medium. After 20 hours, a 1 hour pulse of [.sup.3H]
leucine was given. The cells were then collected onto filters with
a cell harvester. After addition of scintillant, samples were
counted in a Beckman scintillation counter using standard liquid
scintillation counting techniques.
Example 19
Measurement of Cell Viability
In order to measure cell viability of cultures taken at various
time points in fermentation, Ormerod's method was modified
(Ormerod, 2000). Fluorescein diacetate (FDA) and propidium iodide
(PI) were used as vital dyes of cell viability. FDA taken up by
Pichia pastoris was converted to fluorescein by an intracellular
esterase. If a cell has an intact plasma membrane, fluorescein is
retained and PI is excluded. In brief 500 .mu.l of a suspension of
Pichia pastoris cells at 106 cells/ml in the PBS buffer were mixed
with 50 .mu.l of FDA solution (10 .mu.g/ml) and 50 .mu.l of PI
solution (100 .mu.g/ml). After incubation at room temperature for
10 min, cell viability of the sample was analyzed by flow
cytometry. The viable cell gate included green fluorescence and
excluded red fluorescence.
Example 20
Quantification of Concentration of the Bivalent Immunotoxin
A Superdex 200 10/300 GL prepacked column (dimension 1.0
cm.times.30 cm) was purchased from Amersham Pharmacia Biotech. The
column was connected to an HPLC system (GBC Scientific Equipment;
Arlington Heights, Ill., USA). Gel filtration buffer consisted of
90 mM sodium sulfate (Na.sub.2SO.sub.4), 10 mM sodium phosphate
monobasic (NaH.sub.2PO.sub.4.H.sub.2O) and 1 mM EDTA (pH 8.0). The
flow rate was 0.5 ml/min and injection volume was 500 .mu.l.
Purified immunotoxin of known concentration based on UV absorbance
(25) served as a standard.
Quantification of the bivalent immunotoxin in supernatants or
liquid samples, was performed by comparing the intensity of
Coomassie-stained 4-20% precast SDS gels with that of immunotoxin
standards of known concentration.
Example 21
Immunotoxin Toxicity During Expression in Pichia pastoris is
Manifest by a Reduction in AOX1 Activity
The bivalent immunotoxin in Pichia pastoris was expressed via the
secretory route. This secretion of the bivalent immunotoxin in
Pichia pastoris significantly attenuated the toxicity of the
immunotoxin (Woo et al., 2002), but the bivalent immunotoxin
expression depressed metabolic capacity of methanol utilization and
growth reduction during methanol induction in fermentor
culture.
In the metabolism of methanol by Pichia pastoris, oxidation of
methanol by alcohol oxidase (AOX1) is the rate-limiting reaction
(Veenhuis et al., 1983), and the amount of the AOX1 gene product
determines how rapidly methanol is metabolized. AOX1 can account
for 30% of the proteins in Pichia pastoris cells utilizing
methanol. Therefore, measurement of methanol consumption rates
during methanol induction reflects the AOX1 level and provides an
indication of how the expression of the bivalent immunotoxin
affects protein synthesis and degradation of AOX1 in Pichia
pastoris. To this end, profiles of the methanol consumption rate in
a fermentor culture were compared between the wild type host strain
X-33 and the JW102 strain, which expressed the bivalent immunotoxin
via the secretory route. Under the fermentation conditions where
casamino acid supplements were used during methanol induction, X-33
had a maximum 1.95 ml/min of methanol consumption at 25.degree. C.
and the consumption rate was maintained at more than 70% of the
maximum rate during the whole methanol induction phase (FIG. 11).
For the immunotoxin expressing strain JW102 (Mut+), the maximum
methanol consumption rate was approximately 1.10 ml/min at
23.degree. C. After the peak point at 7.about.8 hours following the
initiation of methanol induction, the consumption rate was
gradually decreased to 20% of the maximum rate. Within the first 18
hours of methanol induction, the methanol consumption rate dropped
below 50% of maximum methanol consumption rate (FIG. 11). These low
levels of methanol consumption were associated with low levels of
wet cell density increase, 2% for JW102 versus 10.5% for X-33 at 44
hours (FIG. 14).
Example 22
Use of PMSF and Casamino Acids or Yeast Extract During the Methanol
Induction Phase
In the initial stages of fermentation optimization, supplementing
of PMSF and casamino acids during methanol induction was crucial
for boosting the expression level in the fermentor. Without these
two components during methanol induction, the expression level of
the bivalent immunotoxin reached a maximum 7 hours after initiation
of methanol induction and then decreased. However, supplementing
these two components during methanol induction extended the optimal
induction time from 7.about.8 hours to 24.about.48 hours after the
start of methanol induction. In addition, the expression level was
improved up to 2-fold.
To avoid the use of animal-derived material, yeast extract was
substituted for casamino acids. This change resulted in a
substantial increase in the expression level by 30% and in wet cell
density by 45%. Gain of wet cell density for JW102 by continuous
feeding of yeast extract was close to that for X-33 during methanol
induction (FIG. 14). These improvements were due to constancy of
the methanol consumption rate at greater than 80% of the maximum
rate (FIG. 11).
An example of the final expression method, disclosed herein (see
Example.sub.--41), uses the toxin resistant EF-2 mutant, limited
methanol feeding during induction of 0.5 to 0.75 ml/min (per 10 L
initial medium) without an additional carbon source, extension of
induction time to 163 h, a temperature of 15.degree. C., a
continuous infusion of yeast extract, limitation of agitation speed
to 400 RPM, addition of antifoam agent up to 0.07%, and
supplementation of oxygen when DO levels fall below 40%. Under
these conditions PMSF and Casamino acids are not required. Casamino
acids are an animal product and are frowned upon by the FDA. PMSF,
a protease inhibitor aided to prevent product breakdown, is toxic
and requires additional documentation of its absence from the final
product, so these changes aid regulatory approval. Using this
methodology the yield is 120 mg/L (see example 41).
Example 23
Use of Methanol/Glycerol Mixed Feed During Methanol Induction
The expression level of the bivalent immunotoxin was positively
related to the gain of wet cell density during the first 44 hours
of methanol induction. In low-producing cultures, the gain of wet
cell density was less than 6.0%. However, in fermentation runs
producing more than 25 mg/L of the bivalent immunotoxin, the gain
of wet cell density (%) during the first 44 hours of methanol
induction was an average of 9.26% (FIG. 14). The gain of wet cell
density during methanol induction was hard to achieve without
continuous feeding of glycerol as the additional carbon source.
Therefore a methanol/glycerol (4:1) mixed feed was used to support
cell growth during methanol induction.
Wild type strain X-33 did not produce immunotoxin (FIG. 21A) and
served as a control for monitoring methanol consumption and cell
growth. This strain had a maximum methanol consumption of 1.95
ml/min at 25.degree. C. This consumption rate was maintained at
>70% of the maximum rate for the entire methanol induction phase
(FIG. 21A). The wet cell density increased continuously during the
44 h methanol induction. The DT-resistant immunotoxin producing
EF-2 mutant strain, mutEF2JC307-8(2) was used as another control
for comparing methanol consumption and cell growth upon the
secretion of immunotoxin. This EF-2 mutant strain had similar
profiles of methanol consumption and wet cell growth to those of
wild type strain X-33 during induction (FIG. 21A). The maximum
methanol consumption rate and wet cell gain during 44 h of methanol
induction were 2.2 ml/min and 9.17%, respectively. However, the use
of the EF-2 mutant did not improve immunotoxin secretion under the
fermentation conditions for the JW102 strain producing immunotoxin.
For strain JW102, the maximum methanol consumption rate was 1.30
ml/min at 25.degree. C. After peaking at 7-8 h following the
initiation of methanol induction, the consumption rate decreased to
15% of the maximum rate at 44 h of methanol induction. Within the
first 22 h of methanol induction, the methanol consumption rate
dropped to <50% of the maximum methanol consumption rate (FIG.
21B). This low level of methanol consumption resulted in less
increase in wet cell density, 2.0%, for JW102 than for X-33 (10.5%)
(FIGS. 21A and 21B). There was little or no increase in wet cell
density after the first 22 h of methanol induction and the secreted
level of immunotoxin decreased from 15 to 10 mg/L. Immunotoxin
breakdown products were not detectable on the SDS gels used to
monitor product stability.
Example 24
Yeast Extract Feeding, Methanol Consumption, and Immunotoxin
Production
The decreased methanol consumption and cell growth rate associated
with immunotoxin production can be due to the toxicity of the
immunotoxin to P. pastoris. If yeast extract was fed continuously
to the bioreactor with methanol as the sole carbon source (FIG.
21C), then peak methanol consumption was less than with the wild
type strain and the EF-2 mutant strain (FIG. 21A), but the decrease
after 10 h was eliminated and cell growth increased throughout the
induction period. This growth response was coupled with a loss of
immunotoxin in the medium after 8 h, indicating protease activity.
Immunotoxin fragments were present at 4 h after induction, and no
intact immunotoxin was detected by 19 h after induction (FIG. 22A).
If the medium collected at various time points was incubated with
purified immunotoxin, then the amount of immunotoxin fragments
formed depend on the age of the medium (FIG. 22B). For example, at
49 h post induction the intact immunotoxin band is greatly reduced
and the 36.5 kDa band representing degraded fragments is greatly
increased relative to samples from earlier time points.
The reduction in methanol utilization that was corrected by yeast
extract feeding (FIG. 21C) is apparently secondary to inhibition of
protein synthesis by the immunotoxin following ADP-ribosylation of
EF-2. This was shown by the fact that a P. pastoris strain
producing immunotoxin and engineered to toxin resistance in the
EF-2 gene (13) consumed methanol at the wild type strain rate (FIG.
21 and FIG. 21 legend). In the toxin-sensitive strain, inhibition
of protein synthesis can occur if the immunotoxin gains access to
the cytosol compartment where EF-2 resides. Two distinct mechanisms
can produce this effect. One mechanism is post-translational
translocation where the entire immunotoxin is translated before
entering the Sec61 translocon (16). This would provide a brief
opportunity for ADP ribosylation of EF-2. Post-translational
translocation is common when the signal peptide is alpha mating
factor as it is in this case (24). Another mechanism is the well
documented proton mediated catalytic domain translocation across an
internal membrane compartment (2). This can occur from the mildly
acidic Golgi compartment or the more acidic vacuole. Whichever
immunotoxin translocation mechanism is dominant, yeast extract
feeding either interferes with this step, or with the subsequent
ADP-ribosylation of EF-2 either directly or by attenuating the
catalytic activity of the translocated toxin A chain.
Example 25
Addition of Glycerol to the Methanol Feed with Yeast Extract
Feeding
The protease activity observed when methanol was the sole carbon
source could be a result of leaking from dead or injured cells.
When a 4:1 methanol:glycerol feed (FIG. 21D) was substituted for
the pure methanol (FIG. 21C) the level of immunotoxin in the medium
rose to 20 mg/L at 44 h. Only minimal degradation products now
could be detected in SDS gels of proteins in the medium (FIG. 23,
far right panel). The methanol-glycerol mixed feed without yeast
extract could not sustain the methanol consumption or the continual
increase in cell mass, and the final immunotoxin production was to
15 mg/L (FIG. 21E).
Although continuous feeding of yeast extract largely corrected the
reduction in methanol metabolism, immunotoxin production was low
and was associated with extensive proteolysis (FIGS. 21C and 22).
This extensive proteolysis was reversed by providing supplemental
carbon in the form of a mixed 4:1 methanol:glycerol feed (FIG. 21D
and FIG. 23, panel 23.degree. C.), which increased immunotoxin
production to 20 mg/L. It has been reported that there is an
optimal maximal specific growth rate during P. pastoris methanol
fed-batch culture, which when exceeded depresses heterologous
protein production (27). Feeding methanol at the optimal rate and
adding glycerol at a rate of 20% of the maximal glycerol growth
rate increased heterologous protein production by 50% (27). This
increase can result from increased metabolism of formaldehyde and
H.sub.2O.sub.2 and higher activity of catalase and AOX. In the case
of secreted proteins these metabolic changes also can reduce the
amount of excreted proteases and reduce the number of dead or
injured cells leaking proteolytic enzymes.
Example 26
Low Temperature and Secretion of Bivalent Immunotoxin
Low temperature can improve the yield of heterologous protein
expression in P. pastoris either by enhancing protein folding
within the ER and/or by reducing medium protease activity (9). At
15.degree. C. methanol consumption at 44 h was reduced by 25%,
however cell growth was maintained (FIG. 15C). Immunotoxin
production increased by 50% at 44 h (30.+-.0 mg/L, n=3) and almost
100% at 67 h (37.+-.2.9 mg/L, n=3). Most of the increase in
immunotoxin secretion occurred between 20-15.degree. C.
The highest expression level was observed at 17.5.degree. C. (FIG.
15A), but the final yield obtained by the 3-step purification
procedure of the immunotoxin was the highest at 15.degree. C. (FIG.
15B) and averaged 13.8.+-.1 mg/L (n=3) and 16.0.+-.1 mg/L (n=3) at
44 and 67 h, respectively. The purified immunotoxin produced at
15.degree. C. was fully functional, as confirmed by measuring
specific T cell cytotoxicity in protein synthesis assay yielding
IC50 values for three individual production runs of 1.2
(.+-.0.1).times.10-13 M compared to 2.times.10-13 M for the average
of three runs from shake flask culture.
The amount of degraded immunotoxin bands noted on SDS gels from
bioreactor supernatants (all receiving continuous 10 mM PMSF
feeding) were reduced from modest levels at 23.degree. C. to
undetectable levels at 15.degree. C. (FIG. 23). By using a
sensitive assay for serine Kex-2-like proteases employing a mutant
diphtheria toxin (CRM9) substrate, protease activity was
undetectable at 67 h at 15.degree. C. although activity was
detected at 67 h when PMSF was not infused (FIG. 24). At 15.degree.
C. gel patterns and immunotoxin yields were identical whether or
not PMSF was infused.
Cell viability of 15.degree. C. bioreactor samples from the
methanol-glycerol mixed feed plus yeast extract medium assayed by
flow cytometery had a low level of dead cells: 0.7.+-.0.22%
(confidence limit 99%) glycerol fed-batch phase; glycerol-methanol
mixed feed, 1.2.+-.0.58% (confidence limit 99%) at 22 h,
1.7.+-.0.61% (confidence limit 99%) at 44 h and 1.1.+-.0.51%
(confidence limit 99%) at 67 h (the dead cell fraction was
determined from one fermentation run). The viable cells showing
intracellular esterase activity were present in over 96% of the
cells at all time points during methanol induction.
Lowering the induction temperature from 23-25.degree. C. to
15.degree. C. further increased the immunotoxin level to 30 mg/L at
44 h and 37 mg/L at 67 h (FIG. 21F). Low induction temperature was
associated with a low and constant level of dead cells during
induction (<2.0%) and reduced protease activity toward
immunotoxin within the bioreactor even though small amounts of
protease activity could be detected by a sensitive assay (FIGS. 23
and 24). These results are consistent with a study utilizing
temperature limited (12.degree. C.) fed-batch technique (9). In the
temperature limited fed-batch technique, dead cells were reduced
from 9% to <1% at 44 h compared to a methanol limited fed-batch
process at 30.degree. C. This reduction in dead cells was
associated with a marked reduction in degraded product (lipase) and
a 2-fold increase in intact product at late time points. These
changes were attributed to the avoidance of oxygen deprivation at
high cell densities. AOX activity increased more than 2-fold at 67
h in the temperature-limited fed-batch technique
Lowering induction temperature can also result in increased
immunotoxin secretion by the balancing of immunotoxin input and
output through the secretory pathway by reducing the overall
protein synthesis rate. In the expression and secretion of
heterologous proteins, each protein appears to have an optimal
secretion level. Expression beyond the optimal level
(overexpression) can reduce secreted protein yields (1, 11, 13,
15). The bivalent immunotoxin also can require a longer processing
time for correct folding because of the multi-domain structure of
this protein, which has low activity after in vitro refolding
following expression in E. coli (25). The methanol consumption rate
was reduced by only 25% in going from 23.degree. C. to 15.degree.
C. and the cell growth rate was unchanged at 44 h.
Example 27
Complex Media for Production of Bivalent Immunotoxin in Pichia
pastoris
The uses of the complex components in the initial fermentation
media were necessary to obtain a reasonable range of the expression
level of the bivalent immunotoxin in the fermentor. In the initial
fermentation runs, very low production of the bivalent immunotoxin
in the fermentor was observed when the standard defined medium was
used. Therefore, Soytone Peptone and yeast extract-based medium was
developed containing 4% glycerol, 2% yeast extract, 2% Soytone
Peptone, 1.34% yeast nitrogen base with ammonium sulfate and
without amino acids, 0.43% PTM1 salts solution and 0.01% antifoam
289.
Example 28
Mut+ Versus MutS Phenotype
Different Mut (methanol utilization) phenotype strains derived from
Pichia pastoris GS 115 (Mut+) and KM71 (MutS) were tested to
compare the expression level of the bivalent immunotoxin in the
fermentor. In the fermentor, the MutS phenotype strain has
advantages, such as easy control of induction temperature, no need
to supply pure oxygen, and resistance to a high concentration of
methanol. Although these two different phenotype strains did not
make a difference in the expression level in test tube culture, the
expression level of the Mut+ strain in the fermentor was
5.about.7-fold higher than that of the MutS strain.
Example 29
pH Shifting Procedure Reduces Contaminant Proteins in the
Supernatant
There was a great difference between shake flask culture and
fermentor culture for the expression of the bivalent immunotoxin.
In shake flask culture, it is possible to replace the culture
medium with fresh induction medium, resulting in removal of cell
membrane fragments, DNA and proteases derived from cell lysis
during the growth period and proteins secreted by Pichia pastoris.
However, those molecules accumulate for the whole period of
fermentation and they are often problematic in the purification
process.
In order to reduce this kind of problem in the fermentor, a pH
shifting procedure was employed. Pichia pastoris can normally grow
within the range of pH 3.about.7. Pichia pastoris was cultivated at
a low pH such as pH 3.5 during the glycerol batch phase and the
glycerol-fed batch phase, and induced at pH 7.0 for production of
the bivalent immunotoxin. The pH shifting procedure provided the
supernatant with the dominant bivalent immunotoxin, because the
amount of secreted proteins in Pichia pastoris was significantly
decreased at low pH even though the expression level of the
bivalent immunotoxin was not affected.
Example 30
The Use of Glucose for Tight control of the AOX1 Promoter
In general, tight gene control is necessary to obtain toxic
proteins in host cells. The expression of the bivalent immunotoxin
was toxic to Pichia pastoris. Since the AOX1 promoter cannot
tightly control gene expression in the presence of glycerol as the
carbon source, the bivalent immunotoxin was observed before
methanol induction on Coomassie stained SDS-polyacrylamide gels.
The glycerol-fed batch phase was replaced with a glucose-fed batch
phase for tight gene control, because glucose represses AOX1-driven
gene expression (Tschopp et al., 1987). However, the replacement of
glycerol with glucose in the fed batch phase did not change the
final expression level of the bivalent immunotoxin. Glycerol was
used during the fed batch phase because glucose took time to
dissolve at a high concentration. When combined with the
glycerol-fed batch phase, the pH shifting procedure prevented the
appearance of the bivalent immunotoxin on Coomassie-stained
SDS-polyacrylamide gels during the glycerol-fed batch.
Example 31
Optimal pH for Expression of the Bivalent Immunotoxin
In order to determine optimal pH for the expression of the bivalent
immunotoxin, the expression strain JW102 was induced for 24 hours
in the range of pH 3.5 to 8.0 in test tube cultures, and the
bivalent immunotoxin in the supernatants was compared on a
Coomassie-stained SDS-polyacrylamide gel and Western blotting.
Sodium citrate buffer (pH 3.5.about.5.5), bis-tris buffer (pH
6.0.about.7.0) and tris buffer (pH 7.5.about.8.0) were used for
maintenance of the cultures at the indicated pH. Simultaneously,
colony forming units in the cultures at the end of methanol
induction were measured as previously described (Woo et al., 2002).
Below pH 6.0, the bivalent immunotoxin was not detectable on
Western blots. Although the Western blot shows similar expression
levels in the range of pH 6 to 8, the Coomassie-stained
SDS-polyacrylamide gel indicates pH 7.0 was the optimum pH level
for the expression of the bivalent immunotoxin. Pichia pastoris had
similar colony forming units in the range of pH 3.5 to 7.0, but the
colony forming units were sharply decreased at above pH 7.4. Since
pH 7.4 was the upper edge of optimal pH range, the expression level
at pH 6.7 was also tested in the fermentor. However, there was no
difference in the expression level at pH 6.7 and 7.4.
Example 32
Reproducibility and Cell Viability of Optimized Fermentation
Runs
Under the optimized fermentation conditions, the expression level
of the bivalent immunotoxin increased to 40 mg/L at 67 hours of
methanol induction (FIG. 16). The expression levels in the
supernatants at 44 hours and 67 hours of methanol induction, and
the final yield obtained by the 3-step purification procedure for
the bivalent immunotoxin were reproducible. As shown in Table 4,
very similar levels of bivalent immunotoxin were obtained in 3
independent fermentation runs under the optimized conditions. More
importantly, the final yields of the purified bivalent immunotoxin
were very similar to each other, indicating that produced
supernatants had similar quality of the bivalent immunotoxin. Under
the optimized fermentation conditions, cell viability during
methanol induction phase was maintained at greater than 95% as
determined by flow cytometry.
TABLE-US-00009 TABLE 4 Reproducibility of optimized fermentation
condition.sup.1 and purification.sup.2. Run Methanol induction
Expression Purified immunotoxin no. time (hrs) level (mg/L) from 1
L supernatant (mg) 1 44 30 16 67 40 18 2 44 30 16 67 40 18 3 44 30
16 67 40 18 .sup.1Optimized condition: induction temperature at
15.degree. C.; continuous feeding of 10% yeast extract feeding at
8.95 ml/hr; methanol/glycerol (4:1) mixed feed for methanol
induction. .sup.2For purification of the bivalent immunotoxin, a
3-step procedure (Woo and Neville, 2003) was used.
Example 33
Relationship Between Induction Time and Formation of the
Aggregates
Immunotoxin aggregates were accumulated in the supernatant during
induction. In order to determine the relationship between induction
time and aggregate formation, fractionated samples were taken at
22, 44 and 67 hours of methanol induction by a Superdex 200 gel
filtration and then analyzed fractionated samples on SDS-Page gels.
The 22, 44 and 67 hour samples contained 50.0, 60.0 and 66.7% of
dimeric and higher oligomeric forms of the immunotoxin. These
aggregate forms of the immunotoxin had only 10% specific toxicity
of the monomeric immunotoxin to Jurkat cells.
In addition, the accumulation of immunotoxin aggregates
significantly reduced bioactivity of the supernatant. However,
bioactivity was recovered by the butyl 650M capturing step
developed in a previous study. This result suggested the
possibility that some portion of immunotoxin aggregates were
reversible.
The use of antifoam agents at a concentration above 0.01% reduced
formation of aggregates. These immunotoxin aggregates did not bind
well in thiophilic adsorption used as the capture step before
developing a 3-step purification procedure. In the initial stages
of fermentation optimization, antifoam agents were used at the
minimum concentration that could control excessive foaming in the
fermentor. However, more than 50% of the bivalent immunotoxin was
lost at the first capturing step when antifoam 289 was used at
0.005% in the initial fermentation medium. The use of antifoam 289
at a concentration of more than 0.01% in the initial fermentation
medium was crucial to obtain reasonable yields of more than 90% in
the first capture step.
Example 34
Protein Quantification by Comparison on SDS-PAGE and Cytotoxicity
Assay
The concentration of the immunotoxin was quantified by SDS-PAGE
using an immunotoxin standard of known concentration prepared
previously (Woo et al., 2002). Samples to be measured were
subjected to SDS-PAGE utilizing tris-glycine 4-20% precast gels
(Invitrogen) under non-reducing or reducing conditions.
The specific cytotoxicity of the purified anti-human anti-CD3
immunotoxins were performed as described (Neville et al., 1992).
Briefly, immunotoxins were applied to Jurkat cells, a human
CD3.epsilon.+ T cell leukemia line, (5.times.104 cells/well) in
96-well plates in leucine-free RPMI 1640 medium. After 20 hours, a
1 hour pulse of [3H] leucine was given. Cells were collected onto
filters with a Skatron harvester. After addition of scintillant,
samples were counted in a Beckman scintillation counter using
standard LSC techniques.
Example 35
Butyl 650M Hydrophobic Interaction chromatography (Butyl 650M
HIC)
As shown in FIG. 17, Butyl 650M HIC was an efficient capture step
for immunotoxin in supernatant. However, glycoproteins were also
purified with the immunotoxin during this step. Among these
glycoproteins, identified by periodic acid Schiff staining, the
glycoprotein species of approximately 45 kDa (arrow in FIG. 17)
impeded isolation of the pure immunotoxin. By conventional
chromatography such as gel filtration and anion exchange
chromatography, these glycoproteins were not separated from the
immunotoxin, indicating that these 45 kDa glycoprotein species were
present in dimeric form and had similar isoelectric points.
Therefore these 45 kDa glycoproteins were very similar to the
immunotoxin in size and isoelectric point as well as in
hydrophobicity.
Various hydrophobic resins which complied with GMPs (Good
Manufacturing Practices) were evaluated. Among these resins, Butyl
650M appeared to have the best binding and eluting profile of the
immunotoxin. Other hydrophobic resins may be used in the present
invention. Also it was found that 200 mM of sodium sulfate was a
suitable concentration for binding of the immunotoxin to the butyl
650M resin.
The fermentor culture normally had approximately 30% of wet cell
density at the end of the fermentation run. In large-scale
production, the supernatant is obtained by continuous
centrifugation requiring a 3-fold dilution of the high cell density
culture. The immunotoxin in the diluted sample was processed the
same as the immunotoxin in the supernatant which was effectively
bound to the Butyl 650M resin at 200 mM sodium sulfate.
Example 36
Poros 50 HQ Anion Exchange Chromatography by Step-Eluting with
Sodium Borate Buffer
By employing borate anion exchange chromatography, the immunotoxin
was successfully separated from the Pichia pastoris glycoproteins
(FIG. 18). The immunotoxin was bound to anion resin by diluting the
sample from the previous step, simplifying the purification
procedure. In fractions eluted with 50 mM, 75 mM and 100 mM sodium
borate in Buffer B (lane 9, 10, 11 in FIG. 18), most of the
immunotoxin was present in monomeric form. These 3 fractions were
pooled for the next step.
In order to remove glycoprotein species in the sample obtained from
the previous step, sodium borate in anion exchange chromatography
was used, because sodium borate increases the negative charge of
glycoproteins by binding to the carbohydrate residues of the
glycoproteins. The immunotoxin binds to anion exchange resins at pH
8.0 (Woo et al., 2002). Preliminary experiments were designed for
optimizing binding conditions of the immunotoxin in the presence of
sodium borate. Aliquots of the dialyzed sample against Buffer B
were mixed with different volumes of 200 mM sodium borate in Buffer
B to obtain the designated concentration of sodium borate. The
prepared samples were then loaded onto a Poros 50 HQ anion column
(40 ml) equilibrated with Buffer B containing a corresponding
concentration of sodium borate. At 100 mM of sodium borate the
immunotoxin did not bind to the Poros 50 HQ anion resin, but the
majority of glycoproteins still bound. At a concentration of sodium
borate below 50 mM, the immunotoxin bound to the Poros 50 HQ anion
resin.
Conditions of step elution were further analyzed with sodium borate
after binding of the immunotoxin to an anion exchange column.
First, the sample dialyzed against Buffer B was bound to the anion
column and then eluted in steps of increasing concentration of
sodium borate (100, 120, 140, 200 mM) and 1 M NaCl. The bound
immunotoxin was mainly eluted at 100 mM sodium borate, but these
eluted fractions also contained significant amounts of 45 kDa
glycoproteins which were not separable in the next step. The
majority of glycoproteins were eluted at 1 M NaCl. After loading
the same sample as the first experiment, the bound immunotoxin was
eluted in steps of 50, 75 and 100 mM sodium borate and 1 M NaCl. A
majority of the bound immunotoxin was eluted at 75 mM sodium
borate. However, a protein band corresponding to 21 kDa was
included in the fraction eluted with 50 mM sodium borate. After
binding to the column, the bound immunotoxin was eluted in steps of
25, 50, 75 and 100 mM sodium borate and 1 M NaCl in Buffer B (FIG.
18). By washing with 25 mM sodium borate buffer, the amount of a
protein band corresponding to 21 kDa was reduced.
Example 37
Comparison with Phenylboronate Affinity Chromatography
In order to compare separation profiles, phenylboronate affinity
chromatography was performed. The eluant from the butyl 650M HIC
capture step was dialyzed against the low ionic strength buffer (10
mM HEPES, 0.25 mM EDTA and 20 mM MgCl2, pH 8.2) for phenylboronate
affinity chromatography. The dialysed sample was applied to a 5 ml
bed volume column of phenylboronate agarose (Sigma Co.), washed
with the same buffer, and then the bound proteins were eluted with
either 0-100 mM sodium borate gradient or 0-50 mM sorbitol gradient
in the same buffer (20 bed volumes). Glycoproteins and the
immunotoxin were bound under binding condition of low ionic
strength. The glycoproteins and immunotoxin were not separated by
phenylboronate affinity chromatography. The glycoproteins and
immunotoxin were co-eluted with either 0-100 mM sodium borate
gradient or 0-50 mM sorbitol gradient.
Example 38
Q Anion Exchange Chromatography
Q anion exchange chromatography was used for concentration of the
diluted sample that was obtained from the Poros 50 HQ anion
exchange chromatography. At a concentration of sodium borate below
50 mM the immunotoxin was bound to the anion exchange resin.
Accordingly, the pooled sample from the previous step was diluted
with 3 sample volumes of TE buffer (20 mM Tris-Cl and 1 mM EDTA, pH
8.0), resulting in less than 20 mM of sodium borate in the diluted
sample. As expected, the immunotoxin was effectively bound to the Q
anion exchange column. The bound immunotoxin was eluted with
0.about.400 mM NaCl gradient elution (20 column volumes). The
immunotoxin fractions were pooled and then assessed for yield,
purity and toxicity of the final preparation by SDS-PAGE and
protein synthesis assay.
Example 39
Protein Yield, Repeatability of Purification Procedure, Purity and
Function of the Purified Immunotoxin
Table 4 summarizes the immunotoxin yields which were obtained in 3
batches of the 3-step purification runs by using the supernatants
taken at 44 hours of methanol induction from 3 fermentation runs
which were carried out under relatively similar fermentation
conditions and had similar expression levels of the immunotoxin.
The average yield of this purification batch was 52.8%. By using
the 3-step purification procedure, approximately 16 mg of the
purified material from 1 liter of supernatant was obtained. The
starting supernatants had different levels of immunotoxin
aggregates and monomeric immunotoxin depending on the induction
time during fermentation run. Among these immunotoxin aggregates,
some portions could be reversible to monomeric form of the
immunotoxin during the Butyl 650M HIC step. Fractionation of
supernatant by gel filtration and subsequent SDS-PAGE analysis
showed that the supernatants contained more than 50% of the
immunotoxin aggregates. However, the final yield of the immunotoxin
after the 3-step purification procedure was 52.8%, indicating that
a portion of the aggregates could be dissociated into monomeric
immunotoxin during purification.
A comparison of the purification procedure applied to 3 separate
fermentation runs that contained similar amounts of supernatant
immunotoxin demonstrates good repeatability of the procedure with
respect to yields (Table 5).
The purity of the purified immunotoxin was assessed by analytical
gel filtration. The immunotoxin in the final preparation displayed
a single peak corresponding to the monomeric form of the
immunotoxin (panel A in FIG. 19). The analyses of purity of the
final preparations confirmed that the 3-step purification yielded
an immunotoxin with .about.98.0% purity (panel B in FIG. 19).
To investigate the effects of the 3-step purification procedure on
immunotoxin bioactivity, a protein synthesis assay for the specific
T cell toxicity of the final preparation was performed. The
estimated concentration of the immunotoxin in the final preparation
coincided with concentration of the immunotoxin standard.
TABLE-US-00010 TABLE 5 Comparison of immunotoxin purification from
Pichia pastoris fermentor cultures*. Batch IT conc volume total IT
yield acc. yield no. step (ug/ml) (ml) (mg) (%) (%) 1 Supernatant
30.0 1000 30.0 100.0 100.0 Butyl 650M 45.0 585 26.3 87.8 87.8 HIC
Poros 50 HQ 15.0 1200 18.0 67.0 60.0 borate AEX Q AEX 400.0 40 16.0
88.9 53.3 2 Supernatant 30.0 1000 30.0 100.0 100.0 Butyl 650M 40.0
585 23.4 78.0 78.0 HIC Poros 50 HQ 15.0 1200 17.5 74.6 58.2 borate
AEX Q AEX 400.0 40 16.0 91.6 53.3 3 Supernatant 30.0 1000 30.0
100.0 100.0 Butyl 650M 40.0 585 23.4 78.0 78.0 HIC Poros 50 HQ 15.0
1200 18.0 67.0 60.0 borate AEX Q AEX 450.0 35 15.8 87.5 52.5 *IT,
immunotoxin; acc., accumulated; HIC, hydrophobic interaction
chromatography; AEX, anion exchange chromatography. Supernatants
were obtained from 3 fermentation runs at 44 hours of methanol
induction.
Example 40
Summary
Glycerol feeding decreased immunotoxin proteolysis and enhanced
immunotoxin production while yeast extract feeding primarily
enhanced methanol utilization and cell growth. Glycerol feeding and
yeast extract feeding acted synergistically to increase immunotoxin
production and this synergy was enhanced at 15.degree. C.
This study demonstrates a synergy between carbon source
supplementation with glycerol and continuous yeast extract feeding
that attenuates the toxic effects of the immunotoxin and increases
production, especially at 15.degree. C. This robust process has a
yield of 37 mg/L, 7-fold greater than that previously reported in
the toxin-resistant CHO cell expression system (25).
Example 41
The final method uses the toxin resistant EF-2 mutant, limited
methanol feeding during induction of 0.5 to 0.75 ml/min (per 10 L
initial medium) without an additional carbon source, extension of
induction time to 163 h, a temperature of 15.degree. C., a
continuous infusion of yeast extract, limitation of agitation speed
to 400 RPM, addition of antifoam agent up to 0.07%, and
supplementation of oxygen when DO levels fall below 40%. Under
these conditions PMSF and Casamino acids are not required.
Additionally, reducing shearing force by lowering agitation speed
and adding anti-foam reagent dramatically reduced immunotoxin
aggregation. As a result, purification yield was improved from 64
to 76%. Under this optimized methodology, immunotoxin secretion
level was 120 mg/L at 163 hr of methanol induction. Table 6
summarizes how much immunotoxin secretion and purification yield
were improved by solving the major problems. Gene optimization
enhanced IT secretion from non-detectable level to 10 mg/L. By
using DT-resistant strain and employing low temperature, we
improved immunotoxin secretion up to 35 mg/L. Furthermore,
employing limited methanol feeding improved immunotoxin secretion
as well as purification yield. Finally, extension of induction time
and addition of anti-foam reagent dramatically increased
immunotoxin secretion and purification yield. The anti-foam reagent
is KFOTM 673 which was purchased from Kabo Chemical, Inc.
(Cheyenne, Wyo. 82007, USA). This methodology can be useful for the
production of other recombinant immunotoxins and other toxic
proteins in toxin-sensitive P. pastoris.
TABLE-US-00011 TABLE 6 Increase in immunotoxin secretion and
purification yield by solving major problems IT secretion solutions
level Purification yield Gene optimization 10.0 mg/L n.a. Use of
DT-resistant strain 35.0 mg/L 14.5 mg/L (41.4%) & low
temperature Limited methanol feeding 50.0 mg/L 32.0 mg/L (64.0%)
Extended induction time & 120.0 mg/L 90.8 mg/L (75.7%) addition
of anti-foam reagent
Throughout this application various publications are referenced.
Full citations for these publications are as follow. Such
publications mentioned are hereby incorporated in their entirety by
reference in order to more fully describe the state of the art to
which this invention pertains.
It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
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SEQUENCE LISTINGS
1
35122PRTH. sapiens 1Asp Val Thr Leu His Ala Asp Ala Ile His Arg Gly
Gly Gly Gln Ile 1 5 10 15Ile Pro Thr Ala Arg Arg 20222PRTM.
musculus 2Asp Val Thr Leu His Ala Asp Ala Ile His Arg Gly Gly Gly
Gln Ile 1 5 10 15Ile Pro Thr Ala Arg Arg 20322PRTR. norvegicus 3Asp
Val Thr Leu His Ala Asp Ala Ile His Arg Gly Gly Gly Gln Ile 1 5 10
15Ile Pro Thr Ala Arg Arg 20422PRTC. griseus 4Asp Val Thr Leu His
Ala Asp Ala Ile His Arg Gly Gly Gly Gln Ile 1 5 10 15Ile Pro Thr
Ala Arg Arg 20522PRTD. melanogaster 5Asp Val Thr Leu His Ala Asp
Ala Ile His Arg Gly Gly Gly Gln Ile 1 5 10 15Ile Pro Thr Thr Arg
Arg 20622PRTC. elegans 6Asp Val Thr Leu His Ala Asp Ala Ile His Arg
Gly Gly Gly Gln Ile 1 5 10 15Ile Pro Thr Ala Arg Arg 20722PRTS.
pombe 7Asp Val Val Leu His Ala Asp Ala Ile His Arg Gly Gly Gly Gln
Ile 1 5 10 15Ile Pro Thr Ala Arg Arg 20822PRTP. pastoris 8Asp Val
Thr Leu His Ala Asp Ala Ile His Arg Gly Gly Gly Gln Val 1 5 10
15Ile Pro Thr Met Lys Arg 20922PRTS. cerevisiae 9Asp Val Thr Leu
His Ala Asp Ala Ile His Arg Gly Gly Gly Gln Ile 1 5 10 15Ile Pro
Thr Met Arg Arg 201066DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 10gatgttaccc
tgcacgccga tgctatccac cgccgcggag gacaagtcat tccaaccatg 60aagaga
6611223DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 11actttgaagt tcttaatttt gttcctcgta
gaaagaacgc atagataatt caaaatggca 60aaatgggtat gtgttttttt atagttcatg
tgccgaacaa ctaccgtttt aacttcactg 120tcgatcagat gcgatccctt
atggacaagg tgtccaacgt ccgtaacatg tcggttattg 180cccacgttga
tcacggtaag tccactttaa ctgactccct ggt 22312250DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 12actttgaagt tcttaatttt gttcctcgta gaaagaacgc atagataatt
caaaatgggt 60atgtgttttt ttatagttca tgtgccgaac aactaccgtt tcaagatggg
agccagccac 120taacatctcc tctagttaac ttcactgtcg atcagatgcg
atcccttatg gacaaggtga 180ccaacgtccg taacatgtcg gttattgccc
acgttgatca cggtaagtcc actttaactg 240actccctggt
250132601DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 13atggttaact tcactgtcga tcagatgcga
tcccttatgg acaaggtgac caacgtccgt 60aacatgtcgg ttattgccca cgttgatcac
ggtaagtcca ctttaactga ctccctggtg 120caacgtgccg gtattatttc
tgctgccaag gctggtgagg cccgtttcac tgatactaga 180aaggacgagc
aagagagagg tatcaccatc aagtctaccg ccatttcttt gtactctgag
240atgggtgacg acgatgtcaa ggagatcaag cagaagactg aaggtaacag
tttccttatc 300aacttaattg actccccagg tcacgttgac ttctcttctg
aggtcactgc tgctctgcgt 360gttactgacg gtgctttggt cgtcgttgac
tgtgttgaag gtgtctgtgt tcaaactgag 420accgttttgc gtcaagcttt
gggtgaaaga atcaagccag ttgttgtcat taacaaggtc 480gaccgtgctc
ttttggagtt gcaagttacc aaggaggacc tgtaccagtc tttcgctaga
540accgtcgagt ccgtaaacgt cgttatcgct acttacactg acaagaccat
tggtgacaac 600caagtctacc cagaacaggg taccgtcgct ttcggttcag
gtctgcacgg atgggctttc 660accgttagac agttcgccac tagatactcc
aagaagttcg gtgttgacag aatcaagatg 720atggagcgtc tgtggggaga
ctcttacttc aacccaaaga ccaagaaatg gaccaacaag 780gacaaggacg
ccgctggaaa gcctttggag cgtgccttca acatgttcgt tttggaccct
840atcttccgtc tgtttgctgc catcatgaac ttcaagaagg atgaaattcc
agttctgttg 900gagaaattgg agatcaacct gaagcgtgag gagaaggagt
tggagggtaa ggctcttttg 960aaggttgtca tgagaaagtt cttgccagct
gccgacgctt tgttggagat gattgttctt 1020cacctgccat ctccagtcac
cgctcaagct tacagagccg agactttgta cgaaggtcca 1080tctgatgacc
aattctgcat tggtatcaga gagtgtgacc ctaaggctga gctgatggtt
1140tacatttcca agatggtgcc aacctccgac aaaggtagat tctacgcctt
cggtcgtgtt 1200ttctccggta ctgttaagtc cggtcaaaag gtcagaatcc
aaggtcctaa ctacgttcca 1260ggtaagaagg aggacttgtt catcaaggct
gttcaaagaa ctgttttgat gatgggaaga 1320accgtcgagc ctattgacga
tgtcccagct ggtaacattc tgggtattgt gggtatcgac 1380cagttcttgc
tgaagtctgg tactcttact accaacgaag ccgctcacaa catgaaggtg
1440atgaaattct ctgtctctcc agttgtgcaa gttgccgttg aggtcaagaa
cgctaatgat 1500ctgcccaagt tggttgaggg tctgaagcgt ttgtccaagt
ctgacccatg tgttttaacc 1560tacatctccg agtctggtga gcacattgtt
gctggtactg gtgagctgca cttggaaatc 1620tgtttgcaag atctgcaaga
cgaccacgct ggtgtccctc tgaagatttc tcctccagtt 1680gttacctacc
gtgagactgt cactaacgaa tcttccatga ctgccctgtc caagtctcag
1740aacaagcata acagaattta cctgaaggct caaccaattg acgaggaatt
gtctttggct 1800atcgaagaag gtaaggttca cccaagagac gactttaaag
ccagagccag aatcatggct 1860gatgaatacg gttgggacgt cactgatgcc
agaaagatct ggtgtttcgg tccagacggt 1920actggtgcca acttagttgt
tgaccagtct aaggctgtcc aatacttgca cgagatcaag 1980gactctgttg
ttgccggttt ccaattggct accaaggaag gtccaatttt gggagaaaac
2040atgagatccg tcagagtcaa catcttggat gttaccctgc acgccgatgc
tatccacaga 2100ggtggaggac aagtcattcc aaccatgaag agagttacct
acgccgcctt cctgttggct 2160gagccagcta tccaggagcc tatcttcttg
gtggagatcc aatgtccaga gaatgccatt 2220ggtggtatct actctgtttt
gaacaagaag agaggtcaag ttatctctga ggaacaaaga 2280ccaggtaccc
cattgttcac tgtcaaagct tacttgccag ttaacgagtc attcggtttc
2340accggtgaac tgagacaagc taccgctggt caagctttcc cacagatggt
gttcgaccac 2400tgggccaaca tgaatggtaa cccattggac ccagcctcca
aggtcggtga gattgttctt 2460gctgccagaa agagacaggg tatgaaggag
aacgttcctg gttatgaaga gtactacgac 2520aagttgtaag cttaatgttt
cattaactta tttgtgtcgt tcgtatgtct atttacgtac 2580ttaattcagt
gtattgttgt t 2601149PRTArtificial SequenceDescription of Artificial
Sequence; note = synthetic construct 14Ala His Val Asp His Gly Lys
Ser Thr 1 51513PRTArtificial SequenceDescription of Artificial
Sequence; note = synthetic construct 15Asp Glu Gln Glu Arg Gly Ile
Thr Ile Lys Ser Thr Ala 1 5 1016896PRTArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 16Ala Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val
Met Glu 1 5 10 15Asn Phe Ala Ser Tyr His Gly Thr Lys Pro Gly Tyr
Val Asp Ser Ile 20 25 30Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr
Gln Gly Asn Tyr Asp 35 40 45Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp
Asn Lys Tyr Asp Ala Ala 50 55 60Gly Tyr Ser Val Asp Asn Glu Asn Pro
Leu Ser Gly Lys Ala Gly Gly65 70 75 80Val Val Lys Val Thr Tyr Pro
Gly Leu Thr Lys Val Leu Ala Leu Lys 85 90 95Val Asp Asn Ala Glu Thr
Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105 110Glu Pro Leu Met
Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125Gly Asp
Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135
140Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala
Leu145 150 155 160Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly
Lys Arg Gly Gln 165 170 175Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala
Cys Ala Gly Asn Arg Val 180 185 190Arg Arg Ser Val Gly Ser Ser Leu
Ser Cys Ile Asn Leu Asp Trp Asp 195 200 205Val Ile Arg Asp Lys Thr
Lys Thr Lys Ile Glu Ser Leu Lys Glu His 210 215 220Gly Pro Ile Lys
Asn Lys Met Ser Glu Ser Pro Ala Lys Thr Val Ser225 230 235 240Glu
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 245 250
255Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro
260 265 270Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val
Ala Gln 275 280 285Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys
Thr Thr Ala Ala 290 295 300Leu Ser Ile Leu Pro Gly Ile Gly Ser Val
Met Gly Ile Ala Asp Gly305 310 315 320Ala Val His His Asn Thr Glu
Glu Ile Val Ala Gln Ser Ile Ala Leu 325 330 335Ser Ser Leu Met Val
Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 340 345 350Asp Ile Gly
Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 355 360 365Phe
Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375
380His Lys Thr Gln Pro Phe Leu Pro Trp Asp Ile Gln Met Thr Gln
Thr385 390 395 400Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val
Thr Ile Ser Cys 405 410 415Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu
Asn Trp Tyr Gln Gln Lys 420 425 430Pro Asp Gly Thr Val Lys Leu Leu
Ile Tyr Tyr Thr Ser Arg Leu His 435 440 445Ser Gly Val Pro Ser Lys
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 450 455 460Ser Leu Thr Ile
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe465 470 475 480Cys
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys 485 490
495Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
500 505 510Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys 515 520 525Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe 530 535 540Thr Gly Tyr Thr Met Asn Trp Val Lys Gln
Ser His Gly Lys Asn Leu545 550 555 560Glu Trp Met Gly Leu Ile Asn
Pro Tyr Lys Gly Val Ser Thr Tyr Asn 565 570 575Gln Lys Phe Lys Asp
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 580 585 590Thr Ala Tyr
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val 595 600 605Tyr
Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 610 615
620Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly
Gly625 630 635 640Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Gln Met 645 650 655Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
Leu Gly Asp Arg Val Thr 660 665 670Ile Ser Cys Arg Ala Ser Gln Asp
Ile Arg Asn Tyr Leu Asn Trp Tyr 675 680 685Gln Gln Lys Pro Asp Gly
Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser 690 695 700Arg Leu His Ser
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly705 710 715 720Thr
Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala 725 730
735Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly
740 745 750Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
Gly Gly 755 760 765Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu 770 775 780Leu Val Lys Pro Gly Ala Ser Met Lys Ile
Ser Cys Lys Ala Ser Gly785 790 795 800Tyr Ser Phe Thr Gly Tyr Thr
Met Asn Trp Val Lys Gln Ser His Gly 805 810 815Lys Asn Leu Glu Trp
Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser 820 825 830Thr Tyr Asn
Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 835 840 845Ser
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp 850 855
860Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser
Asp865 870 875 880Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Leu
Thr Val Phe Ser 885 890 8951715DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 17ggggsggggs ggggs
151816DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 18gggsgggsgg gsgggs 16193PRTArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 19Asn Xaa Xaa 12030DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 20ttggttattg
accaaactaa ggctgtccaa 302130DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 21acctctcttc
ttgtttaaga cggagtagat 302239DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 22cttgcttttg
cggccgcttt tttttttttt ttttttttt 392341DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 23gataagaatg cggccgccat ttcttggtct ttgggttgaa g
412442DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 24gataagaatg cggccgccaa cttagttgtt
gaccagtcta ag 422536DNAArtificial SequenceDescription of Artificial
Sequence; note = synthetic construct 25atagctagca ctttgaagtt
cttaattttg ttcctc 362643DNAArtificial SequenceDescription of
Artificial Sequence; note = synthetic construct 26ataagaatgc
ggccgcaagt taatgaaaca ttaagcttac aac 432719DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 27gaatgacttg tcctccacc 192820DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 28gaatgacttg tcctccgcgg 202939DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 29caactagcta gcgctcacaa catgaaggtc atgaaattc
393024DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 30agaaccgtcg agcctattga cgat
243156DNAArtificial SequenceDescription of Artificial Sequence;
note = synthetic construct 31ccctgcacgc cgatgctatc cacagaagag
gaggacaagt cattccaacc atgaag 563221DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 32gccgatgcta tccacagaag a 213321DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 33gccgatgcta tccaccgccg c 213430DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 34tctcttcttg ttcaaaacag agtagatacc 303518DNAArtificial
SequenceDescription of Artificial Sequence; note = synthetic
construct 35gtatgtncac taacntag 18
* * * * *
References