U.S. patent application number 13/327621 was filed with the patent office on 2012-05-24 for recombinant human albumin-granulocyte macrophage colony stimulating factor fusion protein with long-lasting biological effects.
Invention is credited to Yan Fu, Zailin Yu.
Application Number | 20120128624 13/327621 |
Document ID | / |
Family ID | 34192929 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120128624 |
Kind Code |
A1 |
Yu; Zailin ; et al. |
May 24, 2012 |
Recombinant Human Albumin-Granulocyte Macrophage Colony Stimulating
Factor Fusion Protein With Long-Lasting Biological Effects
Abstract
Compositions, kits and methods are provided for promoting
general health or for prevention or treatment of diseases by using
novel recombinant fusion proteins of human serum albumin (HSA) and
bioactive molecules. The bioactive molecules may be a protein or
peptide having a biological function in vitro or in vivo, and
preferably, having a therapeutic activity when administered to a
human. By fusing the bioactive molecule to HSA, stability of the
bioactive molecule in vivo can be improved and the therapeutic
index increased due to reduced toxicity and longer-lasting
therapeutic effects in vivo. In addition, manufacturing processes
are provided for efficient, cost-effective production of these
recombinant proteins in yeast.
Inventors: |
Yu; Zailin; (New York,
NY) ; Fu; Yan; (New York, NY) |
Family ID: |
34192929 |
Appl. No.: |
13/327621 |
Filed: |
December 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12203116 |
Sep 2, 2008 |
8084021 |
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13327621 |
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11825686 |
Jul 8, 2007 |
7442371 |
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12203116 |
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10609346 |
Jun 26, 2003 |
7244833 |
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11825686 |
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60392948 |
Jul 1, 2002 |
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Current U.S.
Class: |
424/85.1 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/348; 435/360;
435/419; 530/363; 536/23.4 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 7/00 20180101; C07K 14/505 20130101; C07K 2319/00 20130101;
C07H 21/04 20130101; Y02A 50/463 20180101; C07K 14/765 20130101;
C07K 14/52 20130101; C07K 14/54 20130101; C07K 14/79 20130101; C07K
14/475 20130101; A61K 47/646 20170801; A61P 37/04 20180101; C07K
14/5431 20130101; A61K 47/643 20170801; A61P 43/00 20180101; C07K
14/5403 20130101; A61K 38/38 20130101; C07K 14/47 20130101; A61K
38/38 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.1 ;
530/363; 536/23.4; 435/320.1; 435/325; 435/348; 435/419; 435/252.3;
435/254.2; 435/360 |
International
Class: |
A61K 38/38 20060101
A61K038/38; C12N 15/62 20060101 C12N015/62; C12N 15/63 20060101
C12N015/63; C12N 1/19 20060101 C12N001/19; C12N 15/85 20060101
C12N015/85; C12N 15/81 20060101 C12N015/81; C12N 5/10 20060101
C12N005/10; C12N 1/21 20060101 C12N001/21; C07K 19/00 20060101
C07K019/00; C12N 15/82 20060101 C12N015/82 |
Claims
1. A human serum albumin-granulocyte macrophage colony stimulating
factor (HSA-GM-CSF) fusion protein comprising: (a) the amino acid
sequence set forth in SEQ ID NO: 10; (b) the amino acid sequence
encoded by the polynucleotide set forth in SEQ ID NO: 9; or (c) the
amino acid sequence encoded by the polynucleotide contained in the
yeast strain designated as YZ-HSA/hGM-CSF in ATCC.RTM. Deposit No:
PTA-4607, said HSA-GM-CSF fusion protein having a plasma half-life
longer than a plasma half-life of granulocyte macrophage colony
stimulating factor.
2. The HSA-GM-CSF fusion protein of claim 1, wherein said
HSA-GM-CSF fusion protein has a shelf-life four times longer than a
shelf-life of the granulocyte macrophage colony stimulating
factor.
3. The HSA-GM-CSF fusion protein of claim 1, wherein said
HSA-GM-CSF fusion protein has said plasma half-life four times
longer than said plasma half-life of the granulocyte macrophage
colony stimulating factor.
4. A composition comprising said HSA-GM-CSF fusion protein of claim
1 and a pharmaceutically acceptable excipient.
5. The composition of claim 4 further comprising a human serum
albumin-cell proliferation stimulatory factor (HSA-CPSF) fusion
protein different from said HSA-GM-CSF fusion protein.
6. A polynucleotide comprising a polynucleotide sequence encoding
said HSA-GM-CSF fusion protein of claim 1.
7. A vector comprising said polynucleotide of claim 6.
8. The vector of claim 7, wherein said vector is an expression
vector for expressing said fusion protein in a host organism
comprising mammal, fish, insect, plant, yeast, or bacterium.
9. The vector of claim 8, wherein said host organism is said
yeast.
10. The vector of claim 9, wherein a strain of said yeast is
selected from the group consisting of Saccharomyces, Candida,
Pichia, Kluyveromyces, Torulaspora, and Schinosaccharomyces.
11. The vector of claim 9, wherein a strain of said yeast is Pichia
pastoris.
12. The vector of claim 7, wherein said vector is a yeast transfer
vector comprising pPICZ A, pPICZ B, or pPICZ C.
13. A host cell comprising said vector of claim 7.
14. The host cell of claim 13, wherein said host cell is selected
from the group consisting of mammalian, fish, insect, plant, yeast,
and bacterial cells.
15. The host cell of claim 14, wherein said mammalian cells are
Chinese hamster ovarian (CHO) cells.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation Application of patent
application Ser. No. 12/203,116, filed Sep. 2, 2008, which is a
Continuation Application of patent application Ser. No. 11/825,686,
filed Jul. 8, 2007, now issued as U.S. Pat. No. 7,442,371 B2, which
is a Continuation Application of patent application Ser. No.
10/609,346, filed Jun. 26, 2003, now issued as U.S. Pat. No.
7,244,833 B2, which claims the priority of Provisional Application
Ser. No. 60/392,948, filed Jul. 1, 2002. All parent applications
are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the manufacture and use of
recombinant albumin fusion proteins and combinations thereof, and
particularly to yeast expressed fusion proteins formed between
human albumin and bioactive molecules such as therapeutic proteins
and peptides, and more particularly to yeast expressed fusion
proteins formed between human albumin and cell proliferation
stimulatory factor (CPSF), such as, blood cell-stimulatory factors,
erythropoietin (EPO), interleukins (ILs), stem cell factor (SCF),
thrombopoietin (TPO), granulocyte colony stimulating factor
(G-CSF), and granulocyte macrophage colony stimulating factor
(GM-CSF).
[0004] 2. Description of Related Art
1. Albumin
[0005] Albumin is a soluble, monomeric protein which comprises
about one-half of the blood serum protein. Albumin functions
primarily as a carrier protein for steroids, fatty acids, and
thyroid hormones and plays a role in stabilizing extracellular
fluid volume. Mutations in this gene on chromosome 4 result in
various anomalous proteins. Albumin is a globular un-glycosylated
serum protein of molecular weight 65,000. The human albumin gene is
16,961 nucleotides long from the putative `cap` site to the first
poly(A) addition site. It is split into 15 exons which are
symmetrically placed within the 3 domains that are thought to have
arisen by triplication of a single primordial domain. Albumin is
synthesized in the liver as pre-pro-albumin which has an N-terminal
peptide that is removed before the nascent protein is released from
the rough endoplasmic reticulum. The product, proalbumin, is in
turn cleaved in the Golgi vesicles to produce the secreted albumin.
HSA has 35 cysteins; in blood this protein monomer has 17 disulfide
linkage (Brown, J. R. "Albumin structure, Function, and Uses"
Pergamon, N.Y., 1977). HSA is misfolded when produced
intracellularly in yeast without its amino terminal secretion
peptide sequence. This conclusion is based on its insolubility,
loss of great than 90% of its antigenicity (as compared to
human-derived HSA), and formation of large protein aggregates. At
present albumin for clinical use is produced by extraction from
human blood. The production of recombinant albumin in
microorganisms has been disclosed in EP 330 451 and EP 361 991.
[0006] Albumin is a stable plasma transporter function provided by
any albumin variant and in particular by human albumin. HSA is
highly polymorphic and more than 30 different genetic alleles have
been reported (Weikamp L, R, et al., Ann. Hum. Genet., 37 219-226,
1973). The albumin molecule, whose three-dimensional structure has
been characterized by X-ray diffraction (Carter D. C. et al.,
Science 244, 1195-1198, 1989), was chosen to provide the stable
transporter function because it is the most abundant plasma protein
(40 g per liter in human), it has a high plasma half-life (14-20
days in human, Waldmann T. A., in "Albumin Structure, Function and
Uses", Rosenoer V. M. et al (eds), Pergamon Press, Oxford,
255-275,1977), and above all it has the advantage of being devoid
of enzymatic function, thus permitting its therapeutic utilization
at high dose.
2. Interleukin-11 (IL-11)
[0007] Human IL-11 (Paul et al. (1990), Pro. Natl. Acad. Sci.
87:7512) has been identified in medium conditioned by primate bone
marrow-derived stromal cells. IL-11 is expressed in cells of
mesenchymal origin, such as stromal fibroblasts, fetal lung
fibroblasts and trophoblasts.
[0008] IL-11, also called adipogenesis inhibitory factor (AGIF),
acts on hematopoietic progenitor cells and stromal cells
(Kawashima, I., et al., Progress in Growth Factor Research, 4,191
1992). The mature molecule is a non-glycosylation protein, 178aa in
length, and has an apparent molecular weight 23 KD (as determined
by SDS-PAGE). Human IL-11 gene consists of five exons and four
introns and was mapped on chromosome 19 at band 19q13.3-q13.4.
IL-11 exhibits a primary structure unrelated to that of known
cytokines, but it often acts similar to other cytokines, notably
IL-6. IL-11 is a pleiotropic growth factor effecting hematopoietic
and non-hematopoietic cells, often in synergy with interleukins,
colony stimulating factors, or stem cell factor. In hematopoietic
cells, IL-11 can enhance megakaryopoiesis, stimulate early and
intermediate myeloid progenitor cells, initiate proliferation of
dormant hematopoietic progenitor cells, and stimulate
T-cell-dependent development of antibody-secreting B-cells. In
non-hematopoietic cells, IL-11 can inhibit adipogenesis, and
mediates the hepatic acute phase response. IL-11 stimulated the
production of erythrocytes was reported only by Quesniaux, VFJ., et
al., Blood, 80, 1218 (1992).
[0009] Human IL-11 (e.g., NEURMEGA.RTM., manufactured by America
Home Products Company) has been approved for clinical trials in the
United States for directly stimulating the proliferation of
hematopoietic stem cells and megakaryocyte progenitor cells and
inducing megakaryocyte maturation, resulting in increased platelet
production. It has been used for the prevention of severe
thrombocytopenia and the reduction of the need for platelet
transfusion following myelosuppressive chemotherapy.
3. Erythropoietin (EPO)
[0010] Erythropoietin (EPO) is a glycoprotein that is the principle
regulator of red blood cells growth and differentiation (U.S. Pat.
No. 5,547,933). Erythropoiesis, the production of red blood cells,
occurs continuously throughout the human life span to offset cell
destruction. Erythropoiesis is a very precisely controlled
physiological mechanism enabling sufficient numbers of red blood
cells to be available in the blood for proper tissue oxygenation,
but not so many that the cells would impede circulation. The
formation of red blood cells occurs in the bone marrow and is under
the control of the hormone EPO.
[0011] EPO is an acidic glycoprotein (.about.30,400 Daltons)
produced primarily by the kidney and is the principal factor
regulating red blood cell production in mammals. Renal production
of EPO is regulated by changes in oxygen availability. Under
conditions of hypoxia, the level of EPO in the circulation
increases and this leads to increased production of red blood
cells. The over-expression of EPO may be associated with certain
pathophysiological conditions. Polycythemia exists when there is an
overproduction of red blood cells (RBCs). Primary polycythemias,
such as Polycythemia vera, are caused by EPO-independent growth of
erythrocytic progenitors from abnormal stem cells and low to normal
levels of EPO are found in the serum of affected patients.
[0012] On the other hand, various types of secondary polycythemias
are associated with the production of higher than normal levels of
EPO. The overproduction of EPO may be an adaptive response
associated with conditions that produce tissue hypoxia, such as
living at high altitude, chronic obstructive pulmonary disease,
cyanotic heart disease, sleep apnea, high-affinity
hemoglobinopathy, smoking, or localized renal hypoxia. In other
instances, excessive EPO levels are the result of production by
neoplastic cells. Cases of increased EPO production and
erythrocytosis have been recorded for patients with renal
carcinomas, benign renal tumors, Wilms' tumors, hepatomas liver
carcinomas, cerebellar hemangioblastomas, adrenal gland tumors,
smooth muscle tumors, and leiomyomas.
[0013] Deficient EPO production is found in conjunction with
certain forms of anemias. These include anemia of renal failure and
end-stage renal disease, anemias of chronic disorders [chronic
infections, autoimmune diseases, rheumatoid arthritis, AIDS,
malignancies], anemia of prematurity, anemia of hypothyroidism, and
anemia of malnutrition. Many of these conditions are associated
with the generation of IL-1 and TNF-, factors that have been shown
to be inhibitors of EPO activity. Other forms of anemias, on the
other hand, are due to EPO-independent causes and affected
individuals show elevated levels of EPO. These forms include
aplastic anemias, iron deficiency anemias, thalassemias,
megaloblastic anemias, pure red cell aplasias, and myelodysplastic
syndromes.
[0014] The amount of erythropoietin in the circulation is increased
under conditions of hypoxia when oxygen transport by blood cells in
the circulation is reduced. Hypoxia may be caused by loss of large
amounts of blood through hemorrhage, destruction of red blood cells
by over-exposure to radiation, reduction in oxygen intake due to
high altitudes or prolonged unconsciousness, or various forms of
anemia. In response to tissues undergoing hypoxic stress,
erythropoietin will increase red blood cell production by
stimulating the conversion of primitive precursor cells in the bone
marrow into proerythroblasts which subsequently mature, synthesize
hemoglobin and are released into the circulation as red blood
cells. When the number of red blood cells in circulation is greater
than needed for normal tissue oxygen requirements, erythropoietin
in circulation is decreased.
[0015] EPO may occur in three forms: alpha-, beta and asialo-EPO.
The alpha- and beta-forms differ slightly in carbohydrate
components, but have the same potency, biological activity and
molecular weight. The asialo-form is an alpha- or beta-form with
the terminal carbohydrate (sialic acid) removed. EPO is present in
very low concentrations in plasma when the body is in a healthy
state wherein tissues receive sufficient oxygenation from the
existing number of erythrocytes. This normal low concentration is
enough to stimulate replacement of red blood cells which are lost
normally through aging. See generally for references, Testa, et
al., Exp. Hematol., 8(Supp. 8), 144-152 (1980); Tong, et al., J.
Biol. Chem., 256(24), 12666-12672 (1981); Goldwasser, J. Cell.
Physiol., 110(Supp 1), 133-135 (1982); Finch, Blood, 60(6),
1241-1246 (1982); Sytowski, et al., Exp. Hematol., 8(Supp 8), 52-64
(1980): Naughton, Ann. Clin. Lab. Sci., 13(5), 432-438 (1983);
Weiss, et al., Am. J. Vet. Res., 44(10), 1832-1835 (1983); Lappin,
et al., Exp. Hematol., 11(7), 661-666 (1983); Baciu, et al., Ann.
N.Y. Acad. Sci., 414, 66-72 (1983); Murphy, et al., Acta.
Haematologica Japonica, 46(7), 1380-1396 (1983); Dessypris, et al.,
Brit. J. Haematol, 56, 295-306 (1984); and, Emmanouel, et al., Am.
J. Physiol., 247 (1 Pt 2), F168-76 (1984).
[0016] Because EPO is essential in the process of red blood cell
formation, the hormone has potential useful application in both the
diagnosis and the treatment of blood disorders characterized by low
or defective red blood cell production. See, generally,
Pennathur-Das, et al., Blood, 63(5), 1168-71 (1984) and Haddy, Am.
Jour. Ped. Hematol./Oncol., 4, 191-196, (1982) relating to
erythropoietin in possible therapies for sickle cell disease, and
Eschbach, et al. J. Clin. Invest., 74(2), pp. 434-441, (1984),
describing a therapeutic regimen for uremic sheep based on in vivo
response to erythropoietin-rich plasma infusions and proposing a
dosage of 10 U EPO/kg per day for 15-40 days as corrective of
anemia of the type associated with chronic renal failure. See also,
Krane, Henry Ford Hosp. Med. J., 31(3), 177-181 (1983).
[0017] Prior attempts to obtain erythropoietin in good yield from
plasma or urine have proven relatively unsuccessful. Complicated
and sophisticated laboratory techniques are necessary and generally
result in the collection of very small amounts of impure and
unstable extracts containing erythropoietin. Genetically engineered
EPO (U.S. Pat. No. 5,547,933) has been expressed in Chinese Hamster
Ovary cell line (CHO). It has been approved for administration on
clinical trials by FDA and now manufactures by Amgen Inc. under the
name of EPOGEN.
[0018] It has been estimated that the availability of
erythropoietin in quantity would allow for treatment each year of
anemias of 1,600,000 persons in the United States alone. See, e.g.,
Morrison, "Bioprocessing in Space--an Overview", pp. 557-571 in The
World Biotech Report 1984, Volume 2:USA, (Online Publications, New
York, N.Y. 1984). Recent studies have provided a basis for
projection of efficacy of erythropoietin therapy in a variety of
disease states, disorders and states of hematologic irregularity:
Vedovato, et al., Acta. Haematol, 71, 211-213 (1984)
(beta-thalassemia); Vichinsky, et al., J. Pediatr., 105(1), 15-21
(1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet.
Gyneacol., 90(4), 304-311 (1983) (pregnancy, menstrual disorders);
Haga, et al., Acta. Pediatr. Scand., 72, 827-831 (1983) (early
anemia of prematurity); Claus-Walker, et al., Arch. Phys. Med.
Rehabil., 65, 370-374 (1984) (spinal cord injury); Dunn, et al.,
Eur. J. Appl. Physiol., 52, 178-182 (1984) (space flight); Miller,
et al., Brit. J. Haematol., 52, 545-590 (1982) (acute blood loss);
Udupa, et al., J. Lab. Clin. Med., 103(4), 574-580 and 581-588
(1984); and Lipschitz, et al., Blood, 63(3), 502-509 (1983)
(aging); and Dainiak, et al., Cancer, 51(6), 1101-1106 (1983) and
Schwartz, et al., Otolaryngol., 109, 269-272 (1983) (various
neoplastic disease states accompanied by abnormal
erythropoiesis).
4. Granulocyte Colony Stimulating Factor (G-CSF)
[0019] Granulocyte colony stimulating factor (G-CSF) is produced by
monocytes and fibroblasts. It stimulating granulocyte colony
formation, activates neutrophils, differentiates certain myeloid
leukemic cell lines and is a potent activator of mature
granulocytes (Metcalf D., cell, 43, 5, 1985; Groopman, J. E.,
Cell., 50, 5 1987);. Nature human G-CSF is a 19.6 KD glycoprotein
having 177 amino acids (Souza, L. M et al., Science, 232, 62,
1986). Human and murine GCSF share approximately 75% amino acid
sequence homology and have biology cross-reactivity (Morstyn, G.,
and Burgess, A., Cancer Res., 48, 5624, 1988). The biological
activity of recombinant human G-CSF was measured in a cell
proliferation assay using NFS-60 cells (Shurafuji, N et al., Exp.
Hematol., 17, 116, 1989). Human G-CSF has been brought to the
market under the name of NEUPOGEN.RTM. by Amgen, Inc.
5. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)
[0020] Granulocyte-Macrophage colony stimulating factor (GM-CSF)
induces myeloid progenitor cells from bone marrow to from colonies
contains macrophages and granulocytes in semisolid media. GM-CSF
also acts upon mature macrophages, eosinophils and nutrophils to
stimulate various functional activities (Mazur, E., and Cohen, J.,
Clin Pharmacol. Ther., 46, 250, 1989; Morstyn, T G., and Burgess.
A., Cancer Res., 48, 5624, 1988). GM-CSF is an acidic glycoprotein
{18-22 KD human (Wong, G., et al., Science, 228, 810, 1986), 23 KD
mouse (Metcalf, D., Blood, 67, 257, 1986)} which binds to high
affinity receptors on GM-CSF sensitive cells. Although human and
mouse GMCSF share 54% amino acid sequence homology, their
biological actions are species-specific (Metcalf, D., Blood, 67,
257, 1986). Other growth factors and CSFs modulate receptor binding
or actions of GM-CSF (Nicola, N., Immunol. Today, 8, 134, 1987).
The proliferative activity of human GMCSF is tested in culture
using human TF-1 cells (Kitamura, T., et al., J. cell Physiol.,
140, 323, 1989). Human GM-CSF has been brought to the market under
the name of LEUKINE.RTM. by Immunex, Inc
6. Macrophage Colony Stimulating Factor (M-CSF)
[0021] Macrophage Colony Stimulating Factor (M-CSF) is produced by
monocytes, fibroblasts and endothelial cells. It stimulates the
formation of macrophage colonies (Metcalf, D., Blood, 67, 257,
1986), enhances antibody-dependent cell mediated cytotoxicity by
monocytes and macrophages (Mufson, R. A. et al., Cellular Immunol.,
119, 182, 1989), and inhibits bone resorption by osteoclasts
(Hattersley, G., et al., J. Cell Physiol., 137, 199, 1988). M-CSF
is glycoprotein and appears in a few different molecular weight
forms due to variation in glycosylation. The peptide has 159 amino
acids (Kawasaki, E. S., et al., Science, 230, 291, 1985).
7. Thrombopoietin (TPO)
[0022] Thrombopoietin (TPO), the ligand for the receptor encoded by
the c-Mpl proto-oncogene, acts as a stimulator of the development
of megakaryocyte precursors of platelets. Similar to
erythropoietin, TPO leads to an increase in number of circulating
platelets. TPO affects the entire thrombopoietic process, with
stronger effects in the later stages. Other thrombopoietic
cytokines include Stem cell factor (SCF), IL-3, IL-6 and IL-11.
[0023] TPO is an approximately 35 KD polypeptide of 335 amino acid.
However, due to glycosylation the protein has an apparent molecular
weight of 75 KD in SDS-PAGE. The precursor form of TPO consists of
356 amino acids. To generate the mature TPO (335aa), the precursor
cleaves a 21 amino acids signal peptide. Human, mouse and dog TPO
shows 69-75% amino acid homology. The biological activities of
recombinant human TPO was measured in a cell proliferation assay
using MO7e cells.
8. Interleukin-3 (IL-3) Interleukin-3 (IL-3) is one of a large and
growing group of growth factors which support the proliferation and
differentiation of hematopoietic progenitors as well as cells
committed to various myeloid lineages in vitro and in vivo. Human
IL-3 has 133 amino acids in mature protein and the glycosylation is
not necessary for biological activity in vitro and in vivo. The
homology between human and murine IL-3 is considerably less. The
initial studies on the biology and biochemistry of IL-3 shows that
among the well characterized hematopoietic growth factors, IL-3, is
the only factor to be predominantly, if not exclusively, produced
by activated T cell in normal cells in mice (Ihele and Weinstein,
1986) as well as in human (Yang and Clark 1998). The structure of
IL-3, and the structure and location of its gene, are very much
like those of a number of the hematopoietic growth factors and
suggest that IL-3 is a member of an evolutionarily related family
of growth factors. In preclinical and clinical trials, the most
prominent and consistent effect of 11-3 in vivo is a significant
increase in the absolute neutrophil count (ANC). In vitro IL-3, in
combination with other cytokines such as stem-cell factor, IL-6,
IL-1, IL-11, G-CSF. GM-CSF, erythropoietin (EPO), or Thrombopoietin
(TPO) induces the proliferation of colony-forming units
granulocyte-macrophage (CFU-GM), CFU-Eo, CFU-Baso, burst-forming
units-erythroid (BFU-E), colony-forming units-megakaryocyte
(CFU-MK) and colony-forming
units-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) in
semisolid medium, and it stimulates the proliferation of purified
CD34+ cells in suspension culture (Eder, et al., Stem Cell,
15:327-333, 1997).
SUMMARY OF THE INVENTION
[0024] The present invention provides compositions, kits and
methods for promoting general health or for prevention or treatment
of diseases by using novel recombinant fusion proteins of human
serum albumin (HSA) and bioactive molecules. The bioactive
molecules may be a protein or peptide having a biological function
in vitro or in vivo, and preferably, having a therapeutic activity
when administered to a human. It is believed that by fusing the
bioactive molecule to HSA, stability of the bioactive molecule in
vivo may be improved and the therapeutic index increased due to
reduced toxicity.
[0025] In one aspect of the invention, recombinant fusion proteins
of human serum albumin (HSA) and a cell proliferation stimulatory
factor (CPSF) are provided 1) to stimulate proliferation of
multiple cell types, especially cells of various developmental
lineages in the blood, 2) allow a slower release of the HSA-CPSF
fusion in the body to maximize the therapeutic effects of the CPSF,
and/or 3) to reduce potential side effects or toxicity associated
with administration of CPSF alone. In addition, manufacturing
processes are provided for efficient, cost-effective production for
producing these recombinant proteins in yeast.
[0026] In another aspect of the invention, an isolated
polynucleotide is provided that encodes a fusion protein formed
between HSA and a CPSF, i.e., an HSA/CPSF fusion. The CPSF may
include any protein that can stimulate cell proliferation and/or
production, preferably selected from the group consisting of
colony-stimulatory factors such as colony-stimulating factors such
as G-CSF, GM-CSF, eosinophil (EOS)-CSF (i.e. Interleukin-5),
macrophage (M)-CSF (CSF-1), multi-CSF(i.e. IL-3) and erythropoietin
(EPO); interleukins such as IL-1; IL-2; IL-4; IL-6; IL-7; IL-9;
IL-10; IL-11; IL-12; IL-13 and IL-18; Steel factors (SLF: c-kit
ligand; Stem-cell factor (SCF); mast cell growth factor); erythroid
potentiating activity (EPA), Lactoferrin (LF), H-subunit ferritin
(i.e., acidic isoferritin), prostaglandin (PG) E1 and E2, tumor
necrosis factor (TNF)-.alpha., -.beta. (i.e. lymphotoxin),
interferon (IFN)-.alpha.(1b, 2a and 2b), -.beta., -.omega. and
-.gamma.; transforming growth factor (TGF)-.beta., activin,
inhibin, leukemic inhibitory factor, oncostatin M; and chemokines
such as macrophage inflammatory protein (MIP)-1-.alpha. (i.e.
Stem-cell inhibitor); macrophage inflammatory protein
(MIP)-1.beta.; macrophage inflammatory protein (MIP)-2-.alpha.
(i.e., GRO-.beta.); GRO-.alpha.; MIP-2-.beta. (i.e., GRO-.gamma.);
platelet factor-4; IL-8; macrophage chemotactic and activating
factor and IP-10.
[0027] The CPSF may be linked directly to the N-terminus or the
C-terminus of HSA to form an HSA-CPSF fusion. Optionally, there is
a peptide linker (L) linking HSA and CPSF together to form the
fusion protein: HSA-L-CPSF, or CPSF-L-HSA. The length of peptide is
preferably between 2-100 aa, more preferably between 5-50 aa, and
most preferably between 14-30 aa. The peptide linker may be a
flexible linker that minimizes steric hindrance imposed by the
bulky HSA protein on CPSF, such as a (G.sub.4S).sub.3-4 linker.
[0028] In one embodiment, an isolated polynucleotide is provided
that encodes a human serum albumin-interleukin-11 fusion protein
(HSA-IL-11). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 1 (FIG. 1). Preferably, the
polynucleotide comprises a nucleotide sequence at least 95%
identical to SEQ ID NO. 1. Preferably, the polynucleotide encodes
an amino acid sequence comprising SEQ ID NO. 2.
[0029] In one embodiment, an isolated polynucleotide is provided
that encodes a human serum albumin-interleukin-3 fusion protein
(HSA-IL-3). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 3. Preferably, the polynucleotide
comprises a nucleotide sequence at least 95% identical to SEQ ID
NO. 3. Preferably, the polynucleotide encodes an amino acid
sequence comprising SEQ ID NO. 4.
[0030] In another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-erythropoietin fusion
protein (HSA-EPO). The polynucleotide comprises a nucleotide
sequence at least 90% identical to SEQ ID NO. 5. Preferably, the
polynucleotide comprises a nucleotide sequence at least 95%
identical to SEQ ID NO. 5. Preferably, the polynucleotide encodes
an amino acid sequence comprising SEQ ID NO. 6. [HSA-EPO].
[0031] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-granulocyte colony
stimulating factor fusion protein (HSA-GCSF). The polynucleotide
comprises a nucleotide sequence at least 90% identical to SEQ ID
NO. 7. Preferably, the polynucleotide comprises a nucleotide
sequence at least 95% identical to SEQ ID NO. 7. Preferably, the
polynucleotide encodes an amino acid sequence comprising SEQ ID NO.
8.
[0032] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-granulocyte macrophage
colony stimulating factor fusion protein (HSA-GMCSF). The
polynucleotide comprises a nucleotide sequence at least 90%
identical to SEQ ID NO. 9. Preferably, the polynucleotide comprises
a nucleotide sequence at least 95% identical to SEQ ID NO. 9.
Preferably, the polynucleotide encodes an amino acid sequence
comprising SEQ ID NO. 10.
[0033] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-CPSF fusion protein
(HSA-CPSF). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 11. Preferably, the
polynucleotide comprises a nucleotide sequence at least 95%
identical to SEQ ID NO. 11. Preferably, the polynucleotide encodes
an amino acid sequence comprising SEQ ID NO. 12. Optionally, the
polynucleotide further comprises a nucleotide sequence at least 90%
identical to SEQ ID NOs. 13, 15, 17, 19, or 21. Preferably, the
polynucleotide further comprises a nucleotide sequence encoding an
amino acid sequence comprising SEQ ID NOs. 14, 16, 18, 20, or
22.
[0034] According to the embodiment, the CPSF may be selected from
the group consisting of G-CSF, GM-CSF, (EOS)-CSF, CSF-1, EPO, IL-1;
IL-2, IL-3, IL-4; IL-6; IL-7; IL-8, IL-9; IL-10; IL-11; IL-12;
IL-13, IL-18, SLF, SCF, mast cell growth factor, EPA, Lactoferrin,
H-subunit ferritin, prostaglandin (PG) E1 and E2, TNF-.alpha.,
TNF-.beta., IFN-.alpha., IFN-.beta., IFN-.alpha.), IFN -.gamma.;
TGF-.beta., activin, inhibin, leukemic inhibitory factor,
oncostatin M, MIP-1-.alpha., MIP-1.beta.; MIP-2-.alpha.,
GRO-.alpha.; MIP-2-.beta., platelet factor-4, macrophage
chemotactic and activating factor and IP-10.
[0035] The fusion protein may be a secretory protein, which binds
to a specific antibody of human albumin, and optionally, binds to a
specific antibody of the CPSF in this fusion protein.
[0036] In another aspect of the invention, a recombinant vector is
provided that comprises the sequence of the polynucleotide
described above. The recombinant vectors can be an expression
vector for expressing the fusion protein encoded by the
polynucleotide, HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSA in a host
organism. The host organism includes, but is not limited to,
mammalian (e.g., human, monkey, mouse, rabbit, etc.), fish, insect,
plant, yeast, and bacterium.
[0037] In a preferred embodiment, the host organism belongs to a
genus of yeast such as Saccharomyces (e.g., S. cerevisiae), Pichia,
Kluyveromyces, Torulaspora, and Schinosaccharomyces. In a more
preferred embodiment, the host organism is Pichia pastoris. In a
particular embodiment, the recombinant vector is a pPICZ A, pPICZ
B, or pPICZ C.
[0038] Depending upon the host organism employed in a recombinant
process for producing the fusion proteins, the recombinant fusion
proteins of the present invention may be glycosylated or may be
non-glycosylated. Preferably, when expressed in a host organism,
the fusion protein of HSA and CPSF is glycosylated to substantially
the same extent as that when expressed in mammalian cells such as
Chinese hamster ovarian (CHO) cells, or as that when expressed in
Pichia pastoris.
[0039] In yet another aspect of the invention, a recombinant cell
is provided that is capable of expressing the sequence of the
polynucleotide described above. The recombinant cell may
constitutively or be induced in the presence or absence of an agent
to express the fusion protein encoded by the nucleic acid,
HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSA in a host organism. The type of
the recombinant cell includes, but is not limited to, mammalian
(e.g., human, monkey, mouse, rabbit, etc.), fish, insect, plant,
yeast, and bacterial cells.
[0040] When stored at ambient temperature or a lower temperature,
the fusion protein of HSA and CPSF may have a shelf-life 2 times
longer, preferably 4 times longer, more preferably 6 times, and
most preferably 10 times, longer than that of the CPSF alone (i.e.,
not fused to HSA) stored under the same condition.
[0041] The fusion protein of the present invention may be
administered to a mammal, preferably a human, via a variety of
routes, including but not limited to, orally, parenterally,
intraperitoneally, intravenously, intraarterially, topically,
transdermally, sublingually, intramuscularly, rectally,
transbuccally, intranasally, liposomally, via inhalation,
vaginally, intraoccularly, via local delivery (for example by
catheter or stent), subcutaneously, intraadiposally,
intraarticularly, or intrathecally. The antibody may also be
delivered to the host locally (e.g., via stents or cathetors)
and/or in a timed-release manner. In a particular embodiment, the
fusion protein is delivered parenterally via injection.
[0042] When delivered in vivo to an animal, the fusion protein of
HSA and CPSF may have a plasma half-life 2 times longer, preferably
4 times longer, more preferably 6 times, and most preferably 10
times, longer than that of the CPSF alone (i.e., not fused to
HSA).
[0043] The HSA/CPSF fusion proteins of the present invention may
also be administered in combination with a natural or recombinant
human albumin, preferably a recombinant human serum albumin at a
therapeutically effective dose and ratio.
[0044] In yet another aspect of the invention, combinations of
different HSA/CPSF fusion proteins of are provided. The specific
combinations of these fusion proteins may be administered to a
patient to stimulate proliferation of multiple types of cells in
the body or to synergistically enhance proliferation of a
particular cell type.
[0045] In one embodiment, HSA/IL-11 fusion may be combined with
HSA/EPO fusion and the resulting combination may be administered to
a patient with a hematological disorder to simultaneously stimulate
proliferation of erythrocytes and platelets.
[0046] In another embodiment, HSA/IL-3 fusion may be combined with
HSA/EPO fusion and the resulting combination may be administered to
a patient with a hematological disorder to enhance EPO-induced
production of erythrocytes.
[0047] In yet another embodiment, HSA/IL-3 fusion may be combined
with HSA/GCSF fusion and the resulting combination may be
administered to a patient with a hematological disorder to increase
the production of erythrocytes and neutrophils, as well as
eosinophils.
[0048] Alternatively, an HSA/CPSF fusion may be co-administered
with a different HSA/CPSF fusion simultaneously or sequentially to
a patient in need thereof. This combination therapy may confer
synergistic therapeutic effects on the patients.
[0049] In yet another aspect of the invention, a method is provided
for treating a patient with a CPSF in need thereof. In one
embodiment, the method comprises: administering a pharmaceutical
formulation comprising a fusion protein of HSA and CPSF to the
patient in a therapeutically effective amount. The pharmaceutical
formulation may contain any pharmaceutically acceptable excipient
and agents that stabilizes the HSA/CPSF fusion protein. The
pharmaceutical formulation may further comprise natural or
recombinant human serum albumin and/or another, different HSA/CPSF
fusion protein.
[0050] The pharmaceutical formulation may contain any
pharmaceutically acceptable excipient and agents that stabilizes
the HSA/CPSF fusion protein. The pharmaceutical formulation may
further comprise natural or recombinant human serum albumin and/or
another, different HSA/CPSF fusion protein.
[0051] In another embodiment, the method comprises: administering a
first pharmaceutical formulation comprising a first fusion protein
of HSA and a first CPSF to the patient in a therapeutically
effective amount; and administering to the patient a second
pharmaceutical formulation comprising a second fusion protein of
HSA and a second CPSF to the patient in a therapeutically effective
amount. Such a combination therapy may confer synergistic
therapeutic effects on the patient.
[0052] For example, HSA-IL-11 fusion protein may be administered to
the patient first, followed by administration of HSA-EPO, HSA-GCSF
and/or HSA-GMCSF at therapeutically effective doses and ratios to
stimulate proliferation of different types of blood cells.
[0053] In yet another aspect of the invention, a kit is provided,
comprising: a first fusion protein of HSA and a first CPSF, and a
second fusion protein of HSA and a second CPSF. The first and
second CPSFs may be the same or different. For example, the first
CPSF is IL-11 and the second CPSF is EPO; the first CPSF is IL-3
and the second CPSF is EPO; or the first CPSF is IL-11 and the
second CPSF is GCSF.
BRIEF DESCRIPTION OF THE FIGURES
[0054] FIG. 1 shows nucleotide and amino acid sequences of
embodiments of HSA-CPSF fusion proteins, HSA, and examples of
individual CPSFs.
[0055] FIG. 2 illustrates a plasmid DNA vector contains the HSA
sequence and as a backbone vector for making HSA-CPSF fusion
proteins.
[0056] FIG. 3 shows a Western blot detected using mouse monoclonal
anti-human serum albumin (Sigma Cat# A6684). Each lane was load
with equivalent of 100 ng of proteins. A), HSA (65 Kd) from blood
plasma; B), HSA (65 Kd) from yeast; C), HSA/hG-CSF (84.2 Kd); D),
HSA/hEPO (83.5 Kd); E), HSA/hIL-11 (84.5 Kd).
[0057] FIG. 4 shows a Western blot detected using goat polyclonal
anti-hIL-11 antibody (R&D Systems, Cat# Ab-218-NA), each lane
contains 100 ng proteins. A), human IL-11 expressed by E. coli; B),
HSA/hIL-11 fusion protein expressed by yeast.
[0058] FIG. 5 shows a Western blot detected using monoclonal
anti-hGMCSF antibody (R&D Systems), each lane contains 20 ng
proteins. A), Human GMCSF expressed by E. coli; B), HSA/hGMCSF
fusion protein expressed in yeast.
[0059] FIG. 6 is a bioassay for human IL-11 and HSA/hIL-11 fusion
protein in stimulation of T1165 cell proliferation. A), with hIL-6
antibody in medium; B), without hIL-6 antibody in medium.
[0060] FIG. 7 is an ELISA bioassay for EPO and HSA fusion protein,
HSA/hEPO.
[0061] FIG. 8 shows the results of a stability test of rHSA/hIL-11
fusion protein under different temperature and its cell
proliferation activity. A), 37.degree. C.; B), 50.degree. C.
[0062] FIG. 9 shows the results of an in vivo animal test of
synergistic effects of a combination of different HSA-CPSFs in
stimulating multiple blood cell proliferation, as compared with
those when single HSA-CPSFs or CPSFs were administered.
[0063] FIG. 10 shows a SDS-PAGE of purified HSA-CPSFs. Each lane
was loaded with about 20 .mu.g of protein. A), HSA (65 Kd) from
blood plasma; B), HSA (65 Kd) from yeast; C), HSA/hG-CSF (84.2 Kd);
D), HSA/hEPO (83.5 Kd); E), HSA/hIL-11(84.5 Kd).
DETAILED DESCRIPTION OF THE INVENTION
[0064] The present invention provides innovative compositions, kits
and methods for modulating cell proliferation in vivo and more
particularly for promoting cell growth for enhancing general health
or for treating diseases or undesirable conditions.
[0065] In general, recombinant fusion proteins of human serum
albumin (HSA) and a cell proliferation stimulatory factor (CPSF)
are provided in order to circumvent problems associated with
conventional therapy using the CPSF protein itself. Generally,
compared with the CPSF protein alone, the inventive fusion proteins
of the present invention possess the following advantages: 1) being
capable of stimulating proliferation of multiple cell types,
especially cells of various developmental lineages in the blood; 2)
allowing a slower release of the HSA-CPSF fusion in the body to
maximize the therapeutic effects of the CPSF, and/or 3) reducing
potential side effects or toxicity associated with administration
of CPSF alone.
[0066] The present invention also provides a method for treating a
patient with a CPSF in need thereof. In one embodiment, the method
comprises: administering a pharmaceutical formulation comprising a
fusion protein of HSA and CPSF to the patient in a therapeutically
effective amount. The pharmaceutical formulation may contain any
pharmaceutically acceptable excipient and agents that stabilizes
the HSA/CPSF fusion protein. The pharmaceutical formulation may
further comprise natural or recombinant human serum albumin and/or
another, different HSA/CPSF fusion protein. The pharmaceutical
formulation may contain any pharmaceutically acceptable excipient
and agents that stabilizes the HSA/CPSF fusion protein. The
pharmaceutical formulation may further comprise natural or
recombinant human serum albumin and/or another, different HSA/CPSF
fusion protein.
[0067] In addition, the present invention also provides
manufacturing processes efficient, cost-effective production for
producing these recombinant fusion proteins in yeast. In
particular, fusion proteins of HSA with each of human IL-11, EPO,
G-GCSF and GM-CSF have been expressed in a yeast strain of Pichia
pastoria and shown to have superior stability in storage and in
plasma, and when combined, to possess synergistic effects on the
production and growth of multiple types of blood cells.
1. HSA/CPSF Fusion Proteins
[0068] In one aspect of the invention, isolated polynucleotides are
provided that encode fusion proteins formed between HSA and a CPSF,
i.e., HSA/CPSF fusion. It should be noted other types of albumin
can also be employed to produce a fusion protein with a CPSF of the
present invention.
[0069] The CPSF may include any protein that can stimulate cell
proliferation and/or production. In a particular embodiment, the
CPSF is a hematopoietically active cytokine Examples of such a CPSF
are described in Aggarwal and Puri (1995) "Role of cytokines in
immuno-regulation", in "Human Cytokines Their role in disease and
therapy", edited by Aggarwal and Puri, Blackwell Science Inc.,
Cambridge, Mass., USA, pp 28, which is incorporated herein by
reference in its entirety.
[0070] Specific examples of the CPSF include, but are not limited
to, colony-stimulating factors such as G-CSF, GM-CSF, eosinophil
(EOS)-CSF (i.e. Interleukin-5), macrophage (M)-CSF (CSF-1),
multi-CSF(i.e. IL-3) and erythropoietin (EPO); interleukins such as
IL-1; IL-2; IL-4; IL-6; IL-7; IL-9; IL-10; IL-11; IL-12; IL-13 and
IL-18; Steel factors (SLF: c-kit ligand; Stem-cell factor (SCF);
mast cell growth factor); erythroid potentiating activity (EPA),
Lactoferrin (LF), H-subunit ferritin (i.e., acidic isoferritin),
prostaglandin (PG) E1 and E2, tumor necrosis factor (TNF)-.alpha.,
-.beta. (i.e. lymphotoxin), interferon (IFN)-.alpha.(1b, 2a, and
2b), -.beta., -.omega. and -.gamma., transforming growth factor
(TGF)-.beta., activin, inhibin, leukemic inhibitory factor,
oncostatin M; and chemokines such as macrophage inflammatory
protein (MIP)-1-.alpha. (i.e. Stem-cell inhibitor); macrophage
inflammatory protein (MIP)-1.beta.; macrophage inflammatory protein
(MIP)-2-.alpha. (i.e., GRO-.beta.); GRO-.alpha.; MIP-2-.beta.
(i.e., GRO-.gamma.); platelet factor-4; IL-8; macrophage
chemotactic and activating factor and IP-10.
[0071] Four distinct colony-stimulating factors (CSFs) that promote
survival proliferation and differentiation of bone marrow precursor
cells have been well characterized: GM-CSF, G-CSF, M-CSF and
Interleukin-3 (IL-3, Multi-CSF). Both GM-CSF and IL-3 are
multipotent growth factors, stimulating proliferation of progenitor
cells from more than one hematopoietic lineage. In contrast, G-CSF
and M-CSF are lineage-restricted hematopoietic growth factors,
stimulating the final mitotic divisions and the terminal cellular
maturation of partially differentiated hematopoietic progenitors.
Erythropoietin is a hematopoietic growth factor that stimulated red
blood cell proliferation. Growth factors, such as Stem Cell Factor,
all the interleukins, and all the interferon, all are CPSF.
[0072] The CPSF may be linked directly to the N-terminus or the
C-terminus of HSA to form an HSA-CPSF fusion. Optionally, there is
a peptide linker (L) linking HSA and CPSF together to form the
fusion protein: HSA-L-CPSF, or CPSF-L-HSA. The length of peptide is
preferably between 2-100 aa, more preferably between 5-50 aa, and
most preferably between 14-30 aa. The peptide linker may be a
flexible linker that minimizes steric hindrance imposed by the bulk
HA protein on CPSF, such as a (G.sub.4S).sub.3-4 linker. The linker
addition may be good for CPSF binds to its receptor.
[0073] The fusion protein may be a secretory protein, which binds
to a specific antibody of human albumin, and optionally, binds to a
specific antibody of the CPSF in this fusion protein.
[0074] In one embodiment, an isolated polynucleotide is provided
that encodes a human serum albumin-interleukin-11 fusion protein
(HSA-IL-11). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 1. Preferably, the polynucleotide
comprises a nucleotide sequence at least 95% identical to SEQ ID
NO. 1. Preferably, the polynucleotide encodes an amino acid
sequence comprising SEQ ID NO. 2. [HSA-IL-11].
[0075] In one embodiment, an isolated polynucleotide is provided
that encodes a human serum albumin-interleukin-3 fusion protein
(HSA-IL-3). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 3. Preferably, the polynucleotide
comprises a nucleotide sequence at least 95% identical to SEQ ID
NO. 3. Preferably, the polynucleotide encodes an amino acid
sequence comprising SEQ ID NO. 4. [HSA-IL-3].
[0076] In another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-erythropoietin fusion
protein (HSA-EPO). The polynucleotide comprises a nucleotide
sequence at least 90% identical to SEQ ID NO. 5. Preferably, the
polynucleotide comprises a nucleotide sequence at least 95%
identical to SEQ ID NO. 5. Preferably, the polynucleotide encodes
an amino acid sequence comprising SEQ ID NO. 6 [HSA-EPO].
[0077] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-granulocyte colony
stimulating factor fusion protein (HSA-GCSF). The polynucleotide
comprises a nucleotide sequence at least 90% identical to SEQ ID
NO. 7. Preferably, the polynucleotide comprises a nucleotide
sequence at least 95% identical to SEQ ID NO. 7. Preferably, the
polynucleotide encodes an amino acid sequence comprising SEQ ID NO.
8 [HSA-GCSF].
[0078] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-granulocyte macrophage
colony stimulating factor fusion protein (HSA-GMCSF). The
polynucleotide comprises a nucleotide sequence at least 90%
identical to SEQ ID NO. 9. Preferably, the polynucleotide comprises
a nucleotide sequence at least 95% identical to SEQ ID NO. 9.
Preferably, the polynucleotide encodes an amino acid sequence
comprising SEQ ID NO. 10 [HSA-GMCSF].
[0079] In yet another embodiment, an isolated polynucleotide is
provided that encodes a human serum albumin-CPSF fusion protein
(HSA-CPSF). The polynucleotide comprises a nucleotide sequence at
least 90% identical to SEQ ID NO. 11. Preferably, the
polynucleotide comprises a nucleotide sequence at least 95%
identical to SEQ ID NO. 11. Preferably, the polynucleotide encodes
an amino acid sequence comprising SEQ ID NO. 12. Optionally, the
polynucleotide further comprises a nucleotide sequence at least 90%
identical to SEQ ID NOs. 13, 15, 17, 19, or 21. Preferably, the
polynucleotide further comprises a nucleotide sequence encoding an
amino acid sequence comprising SEQ ID NOs. 14, 16, 18, 20, or
22.
[0080] According to the embodiment, the CPSF may be selected from
the group consisting of G-CSF, GM-CSF, (EOS)-CSF, CSF-1, EPO, IL-1;
IL-2, IL-3, IL-4; IL-6; IL-7; IL-8, IL-9; IL-10; IL-11; IL-12;
IL-13, IL-18, SLF, SCF, mast cell growth factor, EPA, Lactoferrin,
H-subunit ferritin, prostaglandin (PG) E1 and E2, TNF-.alpha.,
TNF-.beta., IFN-.alpha., IFN-.beta., IFN-.omega., IFN -.gamma.;
TGF-.beta., activin, inhibin, leukemic inhibitory factor,
oncostatin M, MIP-1-.alpha., MIP-1.beta.; MIP-2-.alpha.,
GRO-.alpha.; MIP-2-.beta., platelet factor-4, macrophage
chemotactic and activating factor and IP-10.
[0081] The above-described polynucleotide with a sequence having a
certain degree of sequence identity, for example at least 95%
"identical" to a reference nucleotide sequence encoding a HSA/CPSF
fusion protein, is intended that the polynucleotide sequence is
identical to the reference sequence except that the polynucleotide
sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence encoding the
HSA/CPSF fusion protein. In other words, to obtain a polynucleotide
having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence.
[0082] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the polynucleotide sequence encoding a HSA/CPSF
fusion protein can be determined conventionally using known
computer programs such as the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711).
Bestfit uses the local homology algorithm of Smith and Waterman,
Advances in Applied Mathematics 2:482-489 (1981), to find the best
segment of homology between two sequences. When using Bestfit or
any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are
set, of course, such that the percentage of identity is calculated
over the full length of the reference nucleotide sequence and that
gaps in homology of up to 5% of the total number of nucleotides in
the reference sequence are allowed.
[0083] When stored at ambient temperature or a lower temperature,
the fusion protein of HSA and CPSF may have a shelf-life 2 times
longer, preferably 4 times longer, more preferably 6 times, and
most preferably 10 times, longer than that of the CPSF alone stored
under the same condition.
[0084] The present invention involves the utilization of albumin as
a vehicle to carry a therapeutic protein such as a CPSF that can be
used in the treatment of certain diseases such as cancers, or
people in need of an increased blood cell proliferation in order to
increase the blood cell numbers. The fusion protein of the present
invention may be administered to a mammal, preferably a human, via
a variety of routes, including but not limited to, orally,
parenterally, intraperitoneally, intravenously, intraarterially,
topically, transdermally, sublingually, intramuscularly, rectally,
transbuccally, intranasally, liposomally, via inhalation,
vaginally, intraoccularly, via local delivery (for example by
catheter or stent), subcutaneously, intraadiposally,
intraarticularly, or intrathecally. The HSA-CPSF may also be
delivered to the host locally (e.g., via stents or cathetors)
and/or in a timed-release manner. In a particular embodiment, the
fusion protein is delivered parenterally via injection.
[0085] When delivered in vivo to an animal, the fusion protein of
HSA and CPSF may have a plasma half-life 2 times longer, preferably
4 times longer, more preferably 6 times, and most preferably 10
times, longer than that of the CPSF alone.
[0086] The HSA/CPSF fusion proteins of the present invention may
also be administered in combination with a natural or recombinant
human albumin, preferably a recombinant human serum albumin at a
therapeutically effective dose and ratio.
[0087] It is believed that after fusion with albumin, the CPSF
protein can have a longer shelf-life and plasma half-life, which
allows cost-effective storage and transportation, as well as
reduces amount and/or frequency of drug administration.
[0088] It is noted that the same strategy may be used to develop
stable fusion proteins with anticancer functions. For example,
tumor suppressors, such as p53, pRb, pRb2/p130, and KLF6 may be
fused with HSA. Optionally, insulin and insulin-like growth factor
may be fused with HSA for therapeutic treatment of diabetes. The
gene of antibody from human or other sources with therapeutic
function also can be fused with albumin for easy delivery and
therapeutic purpose. Antigens against which vaccines are developed
can also be fused to albumin for in vivo delivery and increasing
the antigenicity of the antigens. Antibody can be fused to albumin
and then delivered into blood system to induce an immune response
to the invaded foreign antigens such as bacteria, parasites and
viruses. In addition anti-aging proteins may be fused with albumin
and delivered to humans.
2. Expression of Fusion Proteins in Host Organisms
[0089] The polynucleotides encoding the inventive HSA/CPSF fusion
proteins can be cloned by recombinant techniques into vectors which
are introduced to host cells where the fusion proteins can be
expressed.
[0090] Generally, host cells are genetically engineered (transduced
or transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
polynucleotides encoding HSA/CPSF fusion proteins. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0091] According to the invention, a recombinant vector is provided
that comprises the polynucleotide sequence encoding an HSA/CPSF
fusion protein. The recombinant vectors can be an expression vector
for expressing the fusion protein encoded by the nucleic acid,
HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSA in a host organism. The host
organism includes, but is not limited to, mammalian (e.g., human,
monkey, mouse, rabbit, etc.), fish, insect, plant, yeast, and
bacterium.
[0092] Expression of the polynucleotide encoding an HSA/CPSF fusion
protein is under the control of a suitable promoter. Suitable
promoters which may be employed include, but are not limited to,
adenoviral promoters, such as the adenoviral major late promoter;
or heterologous promoters, such as the cytomegalovirus (CMV)
promoter; the respiratory syncytial virus (RSV) promoter; inducible
promoters, such as the MMT promoter, a tetracycline or
tetracycline-like inducible promoter, the metallothionein promoter;
heat shock promoters; the albumin promoter; the ApoAI promoter;
human globin promoters; viral thymidine kinase promoters, such as
the Herpes Simplex thymidine kinase promoter; retroviral LTRs
(including the modified retroviral LTRs hereinabove described); the
.beta.-actin promoter; and human growth hormone promoters. The
promoter also may be the native promoter which controls the
polynucleotide encoding an HSA/CPSF fusion protein.
[0093] Also according to the invention, a recombinant cell is
provided that is capable of expressing comprises the polynucleotide
sequence encoding an HSA/CPSF fusion protein. The recombinant cell
may constitutively or be induced in the presence or absence of an
agent to express the fusion protein encoded by the nucleic acid,
HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSA in a host organism. The type of
the recombinant cell includes, but is not limited to, mammalian
(e.g., human, monkey, mouse, rabbit, etc.), fish, insect, plant,
yeast, and bacterial cell.
[0094] In a preferred embodiment, the host organism belongs to a
genus of yeast such as Saccharomyces (e.g., S. cerevisiae), Pichia,
Kluyveromyces, Torulaspora, and Schinosaccharomyces. In a more
preferred embodiment, the host organism is Pichia pastoris. In a
particular embodiment, the recombinant vector is a pPICZ A, pPICZ
B, or pPICZ C.
[0095] Depending upon the host employed in a recombinant process
for producing the fusion proteins, the fusion proteins of the
present invention may be glycosylated or may be non-glycosylated.
Preferably, when expressed in a host organism, the fusion protein
of HSA and CPSF may be glycosylated to substantially the same
extent as that when expressed in mammalian cells such as Chinese
hamster ovarian (CHO) cells, or as that when expressed in Pichia
pastoris.
[0096] As indicated above, the albumin fusion proteins of the
present invention are substantially preferably proteomic and can
therefore be generated by the techniques of genetic engineering.
The preferred way to obtain these fusion proteins is by the culture
of cells transformed, transfected, or infected by vectors
expressing the fusion protein. In particular, expression vectors
capable of transforming yeasts, especially of the genus Pichia, for
the secretion of proteins will be used.
[0097] It is particularly advantageous to express the HSA/CPSF
fusion protein in yeast. Such an expression system allows for
production of high quantities of the fusion protein in a mature
form, which is secreted into the culture medium, thus facilitating
purification.
[0098] The development of yeast genetic engineering has been made
possible the expression of heterologous genes and the secretion of
their protein products from yeast. The advantages of protein
secretion (export) of yeast are including but not limited to, high
expression level, soluble protein, corrected folding, easy to
scale-up and easy for purification.
[0099] The HSA/CPSF fusion protein can be secreted into the media
of yeast via an albumin natural secretion signal. The polypeptide
sequence of HSA fusion protein can be preceded by a signal sequence
which serves to direct the proteins into the secretory pathway. In
a preferred embodiment the prepro-sequence of human albumin is used
to secret the fusion protein out of yeast cells into the culture
medium. Other secret signal peptides, such as the native
Saccharomyces cerevisiae .alpha.-factor secretion signal, can also
be used to make fusion protein of the present invention.
[0100] Yeast-expressed HSA is soluble and appears to have the same
disulfide linkages as the human-blood derived counterpart. If used
as a pharmaceutical, which may be potentially used in gram amounts
in humans, a recombinant HSA will require a close identity with the
natural HSA product. Secreting the HSA/CPSF fusion protein into the
growth media of yeast, which is via prepro-amino-terminal
processing (no initiator methionine residue), also circumvents the
problems associated with preparing yeast extracts, such as the
resistance of yeast cells to lysis. In addition, the purity of the
product can be increased obtained by placing the product in an
environment in which 0.5-1.0% of total yeast proteins is included
and the lacks toxic proteins that would contaminate the
product.
[0101] In a preferred embodiment, a particular species of yeast
Pichia pastoris is used the system for expressing HSA/CPSF fusions
of the present invention. Pichia pastoris was developed into an
expression system by scientists at Salk Institute
Biotechnology/Industry Association (SIBA) and Phillips Petroleum
for high-level expression of recombinant proteins. The techniques
related to Pichia are taught in, for example, U.S. Pat. Nos.
4,683,293, 4,808,537, and 4,857,467.
[0102] There are some advantages of using yeast Pichia pastoris to
expression of HSA and HSA fusion proteins than using other systems.
Pichia pastoris is a species of yeast genus, Pichia. Pichia has
many of advantages of higher eukaryotic expression systems such as
protein processing, protein folding, and posttranslational
modification, while being as easy to manipulate as E. coli or
Saccharomyces cerevisiae. It is faster, easier, and less expensive
to use than other eukaryotic expression systems such as baculovirus
or mammalian tissue culture, and generally gives higher expression
levels. Pichia has an additional advantage which gives 10 to
100-fold higher heterologous protein expression levels. Those
features make Pichia very useful as a protein expression
system.
[0103] Owing to the similarity between Pichia and Saccharomyces,
many techniques developed for Saccharomyces may be applied to
Pichia. These include: transformation by complementation, gene
disruption, gene replacement. In addition, the genetic nomenclature
used for Sac has been applied to Pichia. For example, histidinol
dehydrogenase is encoded by the HIS4 gene in both Sac and Pichia.
The Pichia as a methylotrophic yeast is capable of metabolizing
methanol as its sole carbon source. The first step in the
metabolism of methanol is oxidation of methanol to formaldehyde
using molecular oxygen by the enzyme alcohol oxidase. In addition
to formaldehyde, this reaction generates hydrogen peroxide. To
avoid hydrogen peroxide toxicity, methanol metabolism takes place
within a specialized cell organelle, called the peroxisome, which
sequesters toxic by-products away from the rest of the cell.
Alcohol oxidase has a poor affinity for O.sub.2, and Pichia
compensates by generating large amounts of this enzyme. The
promoter regulating the production of alcohol oxidase is the one
used to drive heterologous (HSA or HSA fused) protein expression in
Pichia.
[0104] Compared with Saccharomyces cerevisiae, Pichia may have an
advantage in the glycosylation of secreted proteins because it
generally does not hyper-glycosylate. Both Saccharomyces and Pichia
have a majority of N-linked glycosylation of the high-mannose type;
however, the length of the oligosaccharide chains added
post-translation ally to proteins in Pichia (average 8-14 mannose
residues per side chain) is much shorter than those in
Saccharomyces (50-150 mannose residues). Very little O-linked
glycosylation has been observed in Pichia. In addition,
Saccharomyces core oligosaccharide have terminal .alpha.-1,3 glycan
linkages whereas Pichia does not. It is believed that the
.alpha.-1,3 glycan linkages in glycosylated proteins produced from
Saccharomyces are primarily responsible for the hyper-antigenic
nature of those proteins making them particularly unsuitable for
therapeutic use. Although not yet proven, this is predicted to be
less of a problem for glycoprotein generated in Pichia, because it
may resemble the glycoprotein structure of higher eukaryotes.
Protein expressed as a secreted form for correctly refolding and
easy to purification of HSA and HSA fusion proteins. Watanabe, et
al. (2001) "In vitro and in vivo properties of recombinant human
serum albumin from Pichia pastoris purified by a method of short
processing time", Pharm Res 2001 December 18(12):1775; and
Kobayashi, K et al. (1998) "The development of recombinant human
serum albumin" Ther Apher, November 2(4):257-62.
[0105] There are many expression systems available for expressing
in Pichia, such as EasySelect.TM. Pichia Expression Kit from
Invitrogen, Inc. On this vector, an AOX1 promoter is used to allow
methanol-inducible high level expression in Pichia and a Zeocin.TM.
resistance as selective market for the recombinants from the
transformation. Promoters (transcription initiation region) are
very important in expression of fusion proteins in this
invention.
[0106] AOX1 gene promoter is a very strong promoter in yeast
system, especially in Pichia. Two Alcohol Oxidase Proteins are
coded in Pichia for alcohol oxidase--AOX1 and AOX2. The AOX1 gene
is responsible for the vast majority of alcohol oxidase activity in
the cell. Expression of the AOX1 gene is tightly regulated and
induced by methanol to very high levels, typically.gtoreq.30% of
the total soluble protein in cells grown with methanol as the
carbon source. The AOX1 gene has been isolated and a plasmid-bone
version of the AOX1 promoter is used to drive expression of the
gene of interest encoding the desired heterologous protein (Ellis
et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a). While
AOX2 is about 97% homologous to AOX1, growth on methanol is much
slower than with AOX1. This slow growth on methanol allows
isolation of Mut.sup.s strains (aox 1). Except AOX1 gene promoter,
other promoters can also be used to driver HSA fusion gene in
yeast. They are including the promoter from, but not limited to,
PGK1, GAPDH, Gal1, Gal10, CYC1, PH05, TRP1, ADH1, or ADH2 gene.
[0107] The expression plasmid can also take the form of shuttle
vectors between a bacterial host such as E. coli, DH5a from
GIBCO/Life Science and yeast; the antibiotic Zeocin are used to be
a marker for HSA carrier vector in all the examples.
[0108] The expression vector contains the polynucleotide of HSA or
HSA fusion therapeutic protein are introduced into yeast according
to the protocols described in the kit from Invitrogen Inc. After
selection of transformed yeast colonies, those cells expressing the
HSA fusion protein of interest are inoculated into appropriate
selective medium and then tested for their capacity to secrete the
given fusion protein into the extracellular medium. The harvesting
of the protein can be conducted during cell growth for continuous
cultures, or at the end of the growth phase for batch cultures. The
fusion proteins which are the subject of this invention are then
further purified from the culture supernatant by methods which take
into account the albumin purification methods and pharmacological
activities.
[0109] It is noted that other expression systems may also be used
to express rHSA and HSA/CPSF fusion proteins, including but not
limited to E. coli, B. Subtitis, Saccharomyces, Kluyverornyces,
Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces,
Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium,
Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, animals,
plants, and insect cells.
3. Combination Therapy of HSA/CPSF Fusion Proteins
[0110] The present invention also provides combinations of
different HSA/CPSF fusion proteins. The specific combinations of
these fusion proteins may be administered to a patient to stimulate
proliferation of multiple types of cells in the body or to
synergistically enhance proliferation of a particular cell type. In
particular, a combination of human albumin fusions with different
hematopoietically active cytokines is used to effectively promoting
proliferation of the multiple blood cells and platelets. By using a
combination of HSA/CPSF fusion proteins targeting the signal
transduction pathways of different types of blood cells, multiple
blood functional cell production, such as platelets, erythrocytes
and macrophages of white cells, can be increased after
administration by just one injection.
[0111] In the present invention, the albumin's plasma transporter
function and the therapeutic function of the CPSF are integrated
into a fusion form. The presence of albumin may confer a superior
stability to the CPSF by resisting degradation by proteases in the
blood circulation, thus significantly prolonging the plasma half
life of the CPSF. Due to the masking effect of a bulky albumin, a
combination of different CPSFs fused with albumin may impose less
interference with the biological function(s) among different
albumin fused CPSFs than among a combination of the "naked" CPSFs.
Further, a CPSF fused with albumin may be slowly released in the
system over an extensive period of time, thereby reducing the
toxicity associated with injection of the CPSF alone in abnormally
high concentrations in the body. Such a slow release mode of action
of the fusion protein combination can significantly reduce the
amount and/or frequency of injections of the CPSF, thereby further
reducing the side effects of CPSFs. Such combinations are
particularly useful for stimulating multiple blood cell
proliferation after or before the chemo- or radiation therapy of
cancer patients whose tolerance for frequent, high dose injection
of CPSF are seriously compromised.
[0112] For example, in animal testing human TPO, stem cell factor
(SCF) and IL-3 have shown very strong toxicities in vivo. Due to
the severe toxicity in vivo, SCF has been approved by the FDA for
use in vitro only. The limitation on SCF has prevented it from
being developed into useful therapeutics in the clinic. The side
effects of IL-3 are dose-dependent. At a dose higher than 5
.mu.g/Kg, side effects have been observed in patients treated with
IL-3, including fever, rash, fatigue, diarrhea, rigor,
musculoskeletal pain, chills, headache, conjunctivitis, edema,
chest pain, dyspnea, decrease in platelet counts, increasing in
basophilic counts, marrow fibrosis, and pulmonary edema. Eder, M et
al. (1997) "IL-3 in the Clinic", Stem Cells, 15:327-333.
[0113] According to the present invention, HSA fusion protein with
this type of CPSF may remove above limitations by slowly releasing
the drug into the patient's system. In addition, such fusion
proteins may be combined with a relatively higher amount of albumin
to further reduce the impact resulted from directly injecting the
drug into the blood which causes a strong, adverse reaction of the
central nervous system.
[0114] It is also known that "naked" cytokines (i.e., cytokines not
fused to another protein such as HSA) are quite unstable when
stored and have a short plasma half-life. Clearly, a therapeutic
protein with such a weak stability in vivo constitutes a major
handicap. In effect, repeated injections of the product, which are
costly and inconvenient for patient, or an administration of
product by perfusion, become necessary to attain an efficient
concentration in plasma. Due to its extended plasma half-life and
enhanced stability, the HSA/CPSF fusion proteins of the present
invention and their combinations, e.g., HSA fusions with hIL-11,
hEPO, hG-CSF and hGM-CSF, can be used to stimulate the production
of multiple blood cells in plasma of humans.
[0115] In one embodiment, HSA/IL-11 fusion may be combined with
HSA/EPO fusion and the resulting combination may be administered to
a patient with a hematological disorder to simultaneously stimulate
proliferation of erythrocytes and platelets. For example, cancer
patients may be injected with a combination of HSA/IL-11 and
HSA/EPO fusion proteins, before or after, chemotherapy treatment to
avoid blood transfusion and to stimulate proliferation of
erythrocytes and platelets.
[0116] In another embodiment, HSA/IL-3 fusion may be combined with
HSA/EPO fusion and the resulting combination may be administered to
a patient with a hematological disorder to enhance EPO-induced
production of erythrocytes.
[0117] In yet another embodiment, HSA/IL-3 fusion may be combined
with HSA/GCSF fusion and the resulting combination may be
administered to a patient with a hematological disorder to increase
the production of erythrocytes and neutrophils, as well as
eosinophils.
Alternatively, an HSA/CPSF fusion may be co-administered with a
different HSA/CPSF fusion simultaneously or sequentially to a
patient in need thereof. This combination therapy may confer
synergistic therapeutic effects on the patients. In one embodiment,
the method is provided, comprising: administering a first
pharmaceutical formulation comprising a first fusion protein of HSA
and a first CPSF to the patient in a therapeutically effective
amount; and administering to the patient a second pharmaceutical
formulation comprising a second fusion protein of HSA and a second
CPSF to the patient in a therapeutically effective amount. Such a
combination therapy may confer synergistic therapeutic effects on
the patient.
[0118] For example, HSA-IL-11 fusion protein may be administered to
the patient first, followed by administration of HSA-EPO, HSA-GCSF
and/or HSA-GMCSF at therapeutically effective doses and ratios to
stimulate proliferation of different types of blood cells.
[0119] The present invention further provides a kit for use in the
combination therapy described above. The kit comprises: a first
fusion protein of HSA and a first CPSF, and a second fusion protein
of HSA and a second CPSF. The first and second CPSFs may be the
same or different. For example, the first CPSF is IL-11 and the
second CPSF is EPO; the first CPSF is IL-3 and the second CPSF is
EPO; or the first CPSF is IL-11 and the second CPSF is GCSF.
[0120] The HSA/CPSF fusion proteins and their combinations thereof
may be used to treat a wide variety of diseases, including but not
limited to the hematological disorders such as hypochromia,
hypochromic microcytic anemia, and anemia, platelet-less, HIV
infection, cancer, renal failure, and tissue/organ transplantation.
These fusion proteins are preferred not to contain non-human
sequences that may elicit adverse immunogenicity in the
patient.
Examples
1. General Molecular Cloning Techniques
[0121] The classic methods of molecular cloning including, DNA
preparative extractions, agarose and polyacrylamide
electrophoresis, plasmid DNA purification by column or from gel,
DNA fragment ligations, and restriction digestion, are described in
detail in Maniatis T. et al., "Molecular cloning, a Laboratory
Manual", Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y.,
1982 and will not be reiterated here.
[0122] Polymerase Chain Reaction (PCR) used through out all the
examples is described by Saiki, R. K. et al, Science 230:1350-1354,
1985 and is carried out on a DNA thermal cycler (Perkin Elmer)
according to the manufacturer's specification. DNA sequencing was
performed by using standard facilities and following the method
developed by Sanger et al., Proc. Natl. Acad. Sci. USA,
74:5463-5467, 1977. Oligonucleotides were synthesized by commercial
facilities.
[0123] Transformation of E. coli was done by using DH5a competent
cells from GIBCO/BRL. Qiagen plasmid DNA purification columns were
used in the purification of plasmid DNAs. The transformation of
yeast was carried out by electroporation following the instruction
provided by the manufacturer or according to the manual of
EasySelect.TM. Pichia Expression Kit (Invitrogen Inc). All yeast
stains used in the examples are members of the family of Pichia,
and in particular, the strain of Pichia pastoris (supplied by
Invitrogen).
2. Construction of a Backbone Vector Expressing Human Serum
Albumin
[0124] A total RNA isolated from human fetal liver was used in a
reverse transcription polymerase chain reaction (RT-PCR) to
generate the polynucleotide encoding human serum albumin. Briefly,
5 .mu.g of RNA was reverse transcribed by adding a poly(T).sub.18+N
primer and the SuperScript.TM. II RNase H.sup.- reverse
transcriptase (GIBCO/BRL) to make the complementary first strand of
cDNA. The reaction was incubated at 45.degree. C. for 20 minutes,
then at 55.degree. C. for 40 minutes.
[0125] The primers for cloning human serum albumin (HSA) are the
following:
TABLE-US-00001 SEQ ID No. 23: 5'-GAATTCATGAAGTGGGTAACCTTTATTTCC-3'
and SEQ ID No. 24: 5'-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3'.
[0126] These primers were designed based on the HSA sequence
published by GenBank (Access# V00494). Two EcoR I (underline of
primers) sites were created at the 5' end and 3' end for
sub-cloning into an expression vector. After inactivating the
reverse transcriptase at 94.degree. C. for 4 minutes, the DNA
encoding of HSA was further amplified by Taq DNA PCR (Perkin Elmer)
with 35 cycles of 94.degree. C./30 seconds and 58.degree. C./30
seconds and 72.degree. C./2 minutes 30 second, followed by a
72.degree. C./10 minutes incubation. The PCR product (1842 base
pairs) was confirmed by 1% agarose gel electrophoresis. The product
was subcloned into a pCR II TA cloning vector from Invitrogen. DNA
sequencing confirmed that the plasmid DNA contained an insert whose
polynucleotide sequence matches the DNA sequence published in
GenBank (Access# V00494). FIG. 1, Seq ID No. 11 is a polynucleotide
DNA sequence and Seq ID No 12 is the protein amino acid sequence of
human serum albumin.
[0127] After restriction digestion of the PCR product with EcoR I,
the gel purified HSA DNA fragment was inserted into a pPICZ-A
vector (provided by Invitrogen) at the EcoR I site. After
transformation of bacteria DH5a cells with this vector encoding
HSA, a colony was selected from a low salt LB-agar plate contains
25 .mu.g/ml Zeocin. The direction of the insert was confirmed by
restriction enzyme double digestion of plasmid DNA by Xho I/Nde I.
The construct was designated as pYZ-HSA (Y: yeast vector; Z: Zeocin
resistant) and its physical map is shown in FIG. 2.
[0128] There are some advantages associated with the vector
constructed above. It confers resistance to the antibiotic Zeocin.
Zeocin is isolated from Streptomyces and is structurally related to
bleomycin/phleomycin-type antibiotics. Antibiotics in the family of
bleomycin/phleomycin are broad spectrum antibiotics that act as
strong antibacterial and anti-tumor drugs. They show strong
toxicity against bacteria, fungi (including yeast), plants, and
mammalian cells. However, Zeocin is not as toxic as bleomycin on
fungi. A single antibiotic Zeocin could be used in selecting the
recombinants in both bacteria and in yeast. Further, there are
multiple cloning sites at the 3' end of HSA for conveniently
subcloning a CPSF protein in frame to encode a HSA-CPSF. In
addition, a myc epitope sequence and a polyhistidine tag can be
fused to the C-terminal of the expressed fusion protein for easy
detection and/or purification by using commercially available
antibodies against myc or polyhistidine tags. This vector, as a
backbone vector, was used in the construction of expression vectors
for all the HSA fusion proteins described in the Example
section.
3. Molecular Cloning of Human IL-11, EPO, G-CSF, and GM-CSF
3.1. Molecular Cloning Of Human IL-11 Gene
[0129] Human Il-11 was cloned from a total RNA preparation of human
bone marrow-derived stromal cells by RT-PCR method described in
Example 2. The oligonucleotide primers are
TABLE-US-00002 SEQ ID NO. 25: 5'-CATATGAACTGTGTTTGCCGCCTGGTCC-3'
SEQ ID NO. 26: 5'-GATATGTATGACACATTTAATTCCC-3'
[0130] A polynucleotide having 1051 base pairs (bp) was amplified
from RT-PCR reaction and subcloned into pCR II TA cloning vector
from Invitrogen Inc. DNA sequencing confirmed the reading frame of
human IL-11 and inclusion of a 448 bp 3'-end un-translation region
of hIL-11. An Nde I restriction enzyme site was created at the 5'
end. The ATG initiate start codon of hIL-11 was included in this
site (underlined in SEQ ID NO. 25). The DNA sequence of hIL-11 (SEQ
ID NO. 13) and its amino acid sequence (SEQ ID NO. 14) are shown in
FIG. 1.
3.2. Molecular Cloning of Human Erythropoietin Gene
[0131] Human erythropoietin gene was obtained by RT-PCR from human
fetal kidney mRNA from Clontech Laboratory. EPO specific primers
were designed based on the sequence published by Lin, F K et al.,
"Cloning and expression of human erythropoietin gene", Proc. Natl.
Acad. Sci. USA., 82(22):7580-7584 (1985). They are
TABLE-US-00003 SEQ ID NO. 27: 5'-GGATCCATGGGGGTGCACGAATGTCC-3', and
SEQ ID NO. 28: 5'-GAATTCTCATCTGTCCCCTGTCCTGC-3'.
[0132] The PCR product was a full length reading frame of EPO with
two newly created restriction enzymes in each end, Bam HI at the 5'
end and EcoR I at 3' end. The product was inserted into pCR II
vector and sequence confirmed. The human EPO DNA sequence (SEQ ID
NO. 15) and amino acid sequence (SEQ ID NO. 16) are showed in FIG.
1.
3.3. Molecular Cloning of Human G-CSF
[0133] Primers used to clone the human G-CSF gene from a cDNA
library of human fetal liver tissues are
TABLE-US-00004 SEQ ID NO. 29: 5'-GGATCCATGGCTGGACCTGCCACCC-3', and
SEQ ID NO. 30: 5'-GAATTCTCAGGGCTGGGCAAGGTGGC-3'
[0134] These primers were designed based on Nagata, S et al.,
Molecular Cloning and Expression of cDNA for Human Granulocyte
Colony-Stimulating Factor, Nature, 319:415-418, 1986. A Bam HI site
at 5' end and an EcoR I site at 3' end of G-CSF were created. The
PCR products were gel-purified and subcloned into pCR2.1 TA cloning
vectors and DNA sequence was confirmed. The human G-CSF DNA
sequence (SEQ ID NO. 17) and the amino acid sequence (SEQ ID NO.
18) are shown in FIG. 1.
3.4. Molecular Cloning of Human GM-CSF
[0135] Human GM-CSF was cloned from a total RNA sample prepared
from human fetal liver based on Wong, G G et al., "Human GM-CSF:
molecular cloning of the complementary DNA and purification of the
nature and recombinant proteins" Science, 228:810-815, 1985. The
Primers were:
TABLE-US-00005 SEQ ID NO. 31: 5'-GGATCCATGTGGCTGCAGAGCCTGCTGC-3',
and SEQ ID NO. 32: 5'-GAATTCTCACTCCTGGACTGGCTCC-3'
[0136] The PCR products were gel-purified and inserted into pCR2.1
TA cloning vector and sequence confirmed. The human GM-CSF DNA
sequence (SEQ ID NO. 19) and amino acid sequence (SEQ ID NO. 20)
are shown in FIG. 1.
4. In Frame Fusion of HSA With Human IL-11, EPO, G-CSF or
GM-CSF
[0137] There is a Bsu36 I site at the C'-terminus of HSA. All of
the CPSFs described in the Example section were fused into this
site by PCR primer extension to generate a restriction enzyme site
of Bsu36 I at the N-terminus of the CPSF DNA sequence. The CPSF DNA
fragments were amplified by PCR and then subcloned into Bsu36 I and
Xho I sites of pYZ-HSA vector which had been double digested with
Bsu36 I and Xho I to linearize the plasmid DNA
4.1. Construction of Vector Containing Hybrid Polynucleotide of
HSA/hIL-11
[0138] IL-11 gene was fused to HSA C'-terminus by using the
following PCR primers:
TABLE-US-00006 SEQ ID NO. 33:
5'-CTGCCTTAGGCTTACCTGGGCCACCACCTGGCC-3'. (Human IL-11 mature
protein sequence is underlined), and SEQ ID NO. 34:
5'-TGTCGACTCACAGCCGAGTCTTCAGCAGC-3'.
[0139] A Sal I site (underlined in SEQ ID NO. 34) was created at
the 3' end of hIL-11 gene because there is a Xho I site in the
sequence of mature protein of IL-11. The Sal I site sequence is a
cohesive sequence with Xho I site. After ligation, the Sal I and
Xho I site were all gone.
[0140] The PCR products were digested with Bsu36I and Sal I, and
the fragment was gel purified and inserted into pYZ-HSA between of
Bsu36 I and Xho I sites to generate a new plasmid DNA,
pYZ-HSA/hIL-11. The HSA-hIL-11 hybrid polynucleotide sequence (SEQ
ID NO. 1) and its fusion protein amino acid sequence (SEQ ID NO. 2)
are showed in FIG. 1.
4.2. Construction of Vector Containing Hybrid Polynucleotide of
HSA/EPO
[0141] To make a HSA-EPO fusion protein, the following primers were
designed
TABLE-US-00007 SEQ ID NO. 35:
5'-CTGCCTTAGGCTTAATCTGTGACAGCCGAGTCC-3' (human EPO mature protein
sequence underlined), and SEQ ID NO. 36:
5'-CACTCGAGTCATCTGTCCCCTGTCCTGC-3' (Xho I site underlined)
and used to generate the modified human IL-11 DNA fragment. The PCR
products were inserted between Bsu36I and Xho I sites of pYZ-HSA to
generate a pYZ-HSA/hEPO. The HSA-EPO hybrid polynucleotide sequence
(SEQ ID NO. 5) and its fusion protein amino acid sequence (SEQ ID
NO. 6) are shown in FIG. 1.
4.3. Construction of Vector Containing Hybrid Polynucleotide of
HSA/HG-CSF
[0142] Human G-CSF gene was fused with HSA DNA sequence by using
two primers:
TABLE-US-00008 SEQ ID NO. 37:
5'-CTGCCTTAGGCTTAACCCCCCTGGGCCCTGCCAGC-3' (G-CSF mature protein
sequence underlined), and SEQ ID NO. 38:
5'-CTCGAGTCAGGGCTGGGCAAGGTGG-3' (Xho I site at the 3'-teminus of
G-CSF underlined).
[0143] The PCR products were gel purified and subcloned between
Bsu36I and Xho I sites of pYZ-HSA to generate a pYZ-HSA/hG-CSF. The
HSA-G-CSF hybrid polynucleotide sequence (SEQ ID NO. 7) and its
amino acid sequence (SEQ ID NO. 8) are shown in FIG. 1.
4.4. Construction of Vector Containing Hybrid Polynucleotide of
HSA/HGM-CSF
[0144] The following primers:
TABLE-US-00009 SEQ ID NO. 39:
5'-ACTCCTTAGGCTTAGCACCCGCCCGCTCGCCCAGC-3' (GM-CSF mature protein
sequence underlined), and SEQ ID NO. 40:
5'-CTCGAGTCACTCCTGGACTGGCTCC-3' (Xho I site underlined)
[0145] were used to modify GM-CSF DNA sequence in order to subclone
it into pYZ-HSA vector. PCR products were gel purified and double
digested with Bsu36 I and Xho I and inserted between Bsu36 I and
XhoI sites of pYZ-HSA to generate a pYZ-HSA/hGMCSF. The HSA/GM-CSF
hybrid polynucleotide sequence (SEQ ID NO. 9) and its fusion
protein amino acid sequence (SEQ ID NO. 10) are shown in FIG.
1.
5. Transformation of Yeasts
[0146] A yeast Pichia pastoris strain, GS115, colony was inoculated
into 5 ml of YPD medium in a 50 ml conical tube at 30.degree. C.
overnight with shaking at 250 rpm. 0.2 ml of the culture was
inoculated into 500 ml of YPD medium continually shaking at
30.degree. C. for further 2-3 hours or until the cell density reach
to OD.sub.600=1.3-1.5. The cells were collected by centrifuging.
The cell pellet resuspend in 500 ml of ice-cold sterile water in
order to wash the cells. After two rounds water washing, the cells
were resuspended in 20 ml of ice-cold 1M sorbitol to wash again.
The cells finally suspended in 1 ml of ice-cold 1M sorbitol. The
plasmid DNA constructs from Example 2, pYZ-HSA and in Example 4,
pYZ-HSA/IL-11, pYZ-HSA/hEPO, pYZ HSA/hG-CSF, and pYZ-HSA/hGM-CSF
was linearized by Pmel restriction enzyme digestion first.
[0147] Five .mu.g of each linear plasmid DNA was used to transform
80 .mu.l of the freshly made yeast cells in an ice-cold 0.2 cm
electroporation cuvette. The cells mixed with plasmid DNA were
pulsed for 5-10 ms with field strength of 7500V/cm. After the
pulse, 1 ml of ice-cold 1M sorbitol was immediately added into the
cuvette and the content was transferred to a sterile 15 ml tube.
The transformed cells were incubated in 30.degree. C. without
shaking for 2 hours then spread on pre-made YPD-agar plates with
100 .mu.g/ml Zeocin. The colonies were identified with the insert
and the expression level was determined by SDS-PAGE or western-blot
with proper antibodies.
[0148] A consortium of 4 yeast strains produced above (collected
referred to as YZ-HSA/CPSFs) of Pichia pastoris encoding HSA/IL-11,
HSA/hEPO, HSA/hG-CSF, and HSA/hGM-CSF was deposited to the
ATCC.RTM. (America Type Culture Collection) under the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. The 4 yeast
strains are separately designated as YZ-HSA/IL-11, YZ-HSA/hEPO,
YZ-HSA/hG-CSF, and YZ-HSA/hGM-CSF, respectively, and their
consortium is deposited at the ATCC.RTM. under Patent Deposit
Designation No: PTA-4607.
[0149] Different strains of Pichia, such as X-33, KM71 and
proteinase deficient strains SMD1168 and ZY101 were tested for the
expression and secretory of recombinant proteins.
6. Secretion and Characterization of HSA-CPSF Fusion Proteins
Expressed by Pichia
[0150] Several colonies from each transformation of the HSA-CPSF
were cultured with Zeocin in the buffered minimal medium with
glycerol overnight or until OD.sub.600=2-6 at 30.degree. C. and
shaking at 300 rpm. The cultured cells were collected by centrifuge
at 1500 rpm for 5 minutes. Resuspend the cells into buffered
minimal medium without glycerol and cell densities was keep in
OD.sub.600=1.0. 100% methanol was added into each flask to a final
concentration at 0.5% every 24 hours to induce the protein
expression. The culture medium was collected at different time
points and the expression of each fusion protein was confirmed by
SDS-PAGE and western blot. The results showed that human albumin
and HSA-CPSF fusion protein were expressed and secreted into the
medium.
[0151] Mouse monoclonal anti-human serum albumin (Sigma) was used
for immunoblotting on a SDS-PAGE gel. A typical Western blot
experiment was carried on by electrophoresis transfer the protein
from SDS-PAG to a nylon or nitrocellulose filter and incubated with
a specific antibody (as the "first antibody"). Then an anti-first
antibody was added to bind to the first antibody (as the "second
antibody"). The second antibody was labeled with Fluorescence and
the filter was exposed to an X-ray film. Protein molecular weight
standard was used to determine the protein size. The results (FIG.
3) showed that the expressed recombinant proteins, HSA, HSA-CPSF
therapeutic fusion protein, had an expected molecular weight and
also had the same antigen as that of HSA prepared from a human
blood plasma (Sigma). Using monoclonal anti-hIL-11 specific
antibody as the first antibody, the HSA/hIL-11 fusion protein and
human IL-11 (R&D System) had the same antigen and showed that
the molar ratio of HSA to hIL-11 in the HSA/hIL-11 fusion protein
is as expected (FIG. 4). Using monoclonal anti-hGMCSF specific
antibody (R&D System) as the first antibody, the HSA-GMCSF
fusion protein and human GMCSF (R&D System) had the same
antigen and showed that the molar ratio of HSA to GMCSF in the
HSA/GMCSF fusion protein is as expected (FIG. 5).
7. Purification and Molecular Characterization of HSA-CPSF
[0152] The cell culture medium (supernatant) containing the
secreted protein of HSA or HSA-CPSF fusion protein produced from
the recombinant Pichia was collected, the salt concentration
reduced, and the pH was adjusted to above 7.5. The concentrated
sample was passed through an Affi-Gel Blue-gel (50-100 mesh)
(Bio-Rad). The albumin or albumin fusion protein was bound to the
matrix and eluded by a gradient 1-5M NaCl. 75-85% pure albumin or
albumin-CPSF can be recovered in this step. If further purification
is necessary, a size exclusion chromatography is applied to give a
95-99% purity of proteins. The pyrogen was removed from the protein
samples in order to meet the requirement for use in in vivo test.
The Affi-Prep Polymyxin Support (BIO-Rad) column was used to remove
endotoxin from the samples. The purified protein finally passed
through 0.2 .mu.M filter to be sterilized and the protein
concentration was measured by a standard method by using a Bio-Rad
Protein Assay Kit.
8. Cell Proliferation Assay of Human IL-11
[0153] A murine plasmacytoma cell line T1165 which is an IL-6
depended cell line was used to carry out a bioassay for IL-11
according to Paul, S R et al., PNAS 87:7512-7516, 1990. The cells
were maintained in RPMI medium supplemented with 10%
heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (100
U/ml) and streptomycin (100 .mu.g/ml) (Gibco/BRL), 50 .mu.M
2-meracaptoethanol (sigma), and recombinant human IL-6 (4-10 ng/ml
or 20 U/ml in final concentration) (Gibco/BRL). Bioassay is
preformed by 1.times.10.sup.4 cells/well were placed into 96-well
tissue culture plate in 200 ul of IL-6 free medium for 48 hr in
37.degree. C. in the presence of multiple dilution of hIL-11
purified from Thioredoxin fusion protein in E. coli or from yeast
purified HSA/IL-11 fusion protein. In the final 6 hours, the cells
were pulse-labeled with 0.5 mCi of [.sup.3H]thymidine (1Ci=37 GBq,
from Dupont) per well. After incubation, the cells were collected
on to a glass filter, washed and assayed on a Beckman scintillation
counter, Neutralizing goat anti-human IL-6 antibody was included to
abrogate the effect of IL-6 as control in the testing of purified
protein samples. The control proteins including with or without HSA
(from human blood preparation), rHSA expressed in yeast (Pichia) in
the lab, Thioredoxin (Trx) was a peptide purified from the
enterokinase digested Trx/IL-11 fusion protein. The results are
shown in FIG. 6 (A and B panels). The HSA/hIL-11 bioactivities were
not affected by the presence of antibody of human IL-6. HSA fusion
protein has about 1/3 of cell proliferation activity compared with
that human IL-11 alone in the same amount of protein. Since HSA has
a molecular weight about 3 times higher than that of hIL-11, it can
be inferred that HSA-hIL-11 fusion protein has the same bioactivity
as that of human IL-11 alone.
9. Bioassay of EPO by ELISA
[0154] Enzyme-linked immunosorbent assay (ELISA) kit from R&D
Systems was used for the quantitative determination of
erythropoietin (EPO) concentration and bioactivities comparison
with a commercial EPO sample. The EPO ELISA is based on the
double-antibody sandwich method. Microplate wells, precoated with
monoclonal (murine) antibody specific for human EPO were incubated
with samples or standard. Erythropoietin binds to the immobilized
antibody on the plate. After removing excess protein sample or
standard, wells were incubated with an anti-EPO polyclonal (rabbit)
antibody conjugated to horseradish peroxidase. During the second
incubation, the antibody-enzyme conjugate bound to the immobilized
EPO. Excess conjugate was removed by washing. A chromogen was added
into the wells and oxidized by the enzyme reaction to form a blue
colored complex.
[0155] The reaction was stopped by the addition of acid, which
turned the blue to yellow. The amount of color generated was
directly proportional to the amount of conjugate bound to the EPO
antibody complex, which, in turn, was directly proportional to the
amount of EPO in the protein samples or standard. The absorbance of
this complex was measured and a standard curve was generated by
plotting absorbance versus the concentration of the EPO standards.
The EPO concentration of the unknown sample was determined by
comparing the optical density of the protein samples to the
standard curve. The standards used in this assay were recombinant
human EPO (with kit) calibrated against the Second International
Reference Preparation (67/343), a urine-derived form of human
erythropoietin. Human recombinant EPO expressed in CHO cells was
used as a control to determine the rHSA/EPO bioassay
sensitivity.
[0156] The results showed that in the bioassay hEPO fused to HSA
had 1/10 of the sensitivity compared with the standard (FIG. 7).
The size of HSA-EPO fusion protein molecule may be too large, which
prevents the anti-EPO antibody from efficiently binding to the EPO
molecule fused to HSA, thereby reducing the sensitivity of the
detection in this bioassay.
10. Stability Testing of HSA-CPSF Fusion Proteins In Vitro
[0157] Using HSA/hIL-11 as an example, the stability of this
HSA-CPSF fusion protein was tested at different time points at
37.degree. C. and 50.degree. C. 5 ng of human IL-11 from bacteria
or 5 ng of rHSA/hIL-11 was put into 200 .mu.l thin-well PCR tube
with 200 .mu.l of tissue culture medium RPM1 without fetal bovine
serum and other components. The tubes were sealed and left in water
bath. Samples were taken out at different time points and
immediately put into -80.degree. C. for storage. After all of
samples were collected, a cell proliferation test on T1165 cell
line was carried out by incorporating .sup.3H-Thymidine into newly
synthesized DNA of proliferating cells. The control of the test was
set up in the same way as that in the bioassay of human IL-11 (See
Paul et al., 1990). As shown in FIG. 8, after 5 weeks in 37.degree.
C. (Panel A), the bioactivity of HSA/IL-11 still remained the same,
but the "naked" human IL-11 lost almost all of its bioactivity
after three weeks at 37.degree. C. At 50.degree. C. (Panel B), the
"naked" human IL-11 lost its the bioactivity completely in 2 weeks.
And the HSA/IL-11 fusion protein still retained at least half of
its bioactivity. These results indicate that a CPSF fused to human
albumin can have a longer storage time and more resistant to
degradation in harsh environment such as high temperatures.
11. Synergistic Effects of Combination of HSA-CPSF Fusion Proteins
in Stimulation of Multicell Proliferation
[0158] Rabbits (2.3-2.6 Kg) were injected with 200 .mu.l of
recombinant proteins prepared above at day 1, day 3 and day 6.
Rabbit A was injected with a mixture of 150 U/kg EPO (about 10
.mu.g of protein) and 100 .mu.g recombinant HSA (rHSA); Rabbit B
with a mixture of 120 .mu.g IL-11 and 100 .mu.g rHSA; and Rabbit C
with a mixture of 120 .mu.g rHSA/hIL-11 fusion (equivalent of 50
.mu.g of pure bacteria-expressed IL-11), 27 .mu.g rHSA/hEPO fusion
(equivalent of 150 U of HSA-EPO determined by using the EPO-ELISA
Kit, R&D System Inc.) and 50 .mu.g rHSA. Rabbit D was injected
with 120 .mu.g of HSA-IL-11 and about 80 .mu.g rHSA.
[0159] Blood samples were collected and red blood cell and platelet
numbers were counted by a hemacytometer. The results are shown in
FIG. 9 (panels A, B, and C). The cell counts on the starting day of
the experiment was treated as the base line. After the treatment
with the proteins, the cell counts were compared with those on the
starting day and the changes were plotted in the graphs in FIG.
9.
[0160] As shown in panel A of FIG. 9, both EPO and HSA/EPO fusion
protein stimulated the production of erythrocytes in rabbit A and
C, respectively. However, in rabbit A injected with the naked EPO,
the level of erythrocytes reached a peak around day 35 post first
injection and then declined quickly to reach near a baseline level
around day 55. In contrast, in rabbit C injected with HSA/EPO
fusion protein, the level of erythrocytes increased and reached a
plateau around day 35 post first injection but remained high till
the end of the experiment. These results demonstrated that HSA/EPO
fusion protein has a much longer plasma half-life than the naked
EPO and remains bioactive for a much longer time than the naked EPO
in vivo. As also shown in this panel, IL-11 had little effect in
stimulation of erythrocyte production in rabbit B.
[0161] As shown in panel B of FIG. 9, both IL-11 and HSA/IL-11
fusion protein stimulated the production of platelets in rabbit B
and C, respectively. However, in rabbit B injected with the naked
IL-11, the level of platelets reached a peak around day 20 post
first injection and then declined quickly to reach near a baseline
level around day 43. In contrast, in rabbit C injected with
HSA/IL11 fusion protein, the level of platelets increased and
reached a plateau around day 40 post first injection but remained
high till the end of the experiment. These results demonstrated
that HSA/IL-11 fusion protein has a much longer plasma half-life
than the naked IL-11 and remains bioactive for a much longer time
than the naked IL-11 in vivo. As also shown in this panel, EPO had
little effect in stimulation of platelet production in rabbit
A.
[0162] Panel C in FIG. 9 compares the effects of a combination of
HSA/EPO and HSA/IL-11 with HSA/EPO and HSA/IL-11 individually in
stimulation of the production of erythrocytes and platelets on day
35 post first injection. As shown in this panel, compared with
individual HSA/EPO and HSA/IL-11, a combination of HSA/EPO and
HSA/IL-11 had much stronger effects in stimulating the production
of both erythrocytes and platelets. For example, the erythrocyte
level in the rabbit C which was injected with 27 .mu.g of HSA/EPO
in combination 120 .mu.g HSA/IL-11 with is higher than the total
erythrocyte level combining that in rabbit A (injected with 10
.mu.g HSA/EPO) and rabbit D (120 .mu.g HSA/IL-11). Since the molar
ratio of HSA to EPO in the HSA/EPO fusion is about 5:1 and the
amount of HSA/EPO in rabbit C is only 2.7 folds of that in Rabbit
A, it can be predicted that if the amount of HSA/EPO is increased 5
folds so that a equal mole of HSA/EPO in both the HSA/EPO+HSA/IL-11
combination and HSA/EPO alone is administered, the erythrocyte
level in the animal should be even higher with the administration
of the combination. Based on this analysis, it can be reasonably
inferred that a combination of HSA/EPO+HSA/IL-11 fusion proteins
has a synergistic effect in stimulating multiple blood cell
production.
12. Expression and Scale-Up of HSA-CPSF Fusion Protein by
Fermentation
[0163] In this example, it is shown that expression and scale-up
are much easier by using a Pichia system than other currently
available systems. After Pichia recombinants were isolated,
expression of both Mut+ and Mut.sup.s recombinants was tested. This
involved growing a small culture of each recombinant, inducing with
methanol, and taking sample at different time points. For secretory
expression, both the cell pellet and supernatant were analyzed from
each time point. The samples were analyzed on SDS-PAGE gels by
using both Coomassie staining and Western blot. Bioactivities of
expressed samples were tested and the expression levels and purity
were monitored in each step for production of HSA fusion proteins.
FIG. 10 shows a SDS-PAGE of the purity of various HSA-CPSFs fusion
proteins.
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Sequence CWU 1
1
4012229DNAArtificial SequenceDNA of HSA-hIL-11 1atgaagtggg
taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc
gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa
120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca
gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg
caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt
catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac
ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg
aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg
420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac
atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt
atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca
gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga
tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt
gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta
720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa
gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc
tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa
aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct
gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg
ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc
1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga
atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg
ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct
catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga
gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag
agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc
1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt
gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag
aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa
acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa
caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca
aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt
1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt
gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg
atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc
tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt
aggcttagct cccatgaccc agacaacgtc cttgaagaca 1860agctgggtta
actgctctaa catgatcgat gaaattataa cacacttaaa gcagccacct
1920ttgcctttgc tggacttcaa caacctcaat ggggaagacc aagacattct
gatggaaaat 1980aaccttcgaa ggccaaacct ggaggcattc aacagggctg
tcaagagttt acagaacgca 2040tcagcaattg agagcattct taaaaatctc
ctgccatgtc tgcccctggc cacggccgca 2100cccacgcgac atccaatcca
tatcaaggac ggtgactgga atgaattccg gaggaaactg 2160acgttctatc
tgaaaaccct tgagaatgcg caggctcaac agacgacttt gagcctcgcg
2220atcttttag 22292763PRTArtificial SequenceHSA-hIL-11 2Asp Ala His
Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn
Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40
45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr
Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys
Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425
430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val
Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys
Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser
Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn
Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser
Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu
Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys
Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu
Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu Pro Gly Pro Pro
Pro Gly Pro 580 585 590Pro Arg Val Ser Pro Asp Pro Arg Ala Glu Leu
Asp Ser Thr Val Leu 595 600 605Leu Thr Arg Ser Leu Leu Ala Asp Thr
Arg Gln Leu Ala Ala Gln Leu 610 615 620Arg Asp Lys Phe Pro Ala Asp
Gly Asp His Asn Leu Asp Ser Leu Pro625 630 635 640Thr Leu Ala Met
Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly 645 650 655Val Leu
Thr Arg Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val 660 665
670Gln Trp Leu Arg Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro
675 680 685Glu Leu Gly Thr Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg
Arg Leu 690 695 700Gln Leu Leu Met Ser Arg Leu Ala Leu Pro Gln Pro
Pro Pro Asp Pro705 710 715 720Pro Ala Pro Pro Leu Ala Pro Pro Ser
Ser Ala Trp Gly Gly Ile Arg 725 730 735Ala Ala His Ala Ile Leu Gly
Gly Leu His Leu Thr Leu Asp Trp Ala 740 745 750Val Arg Gly Leu Leu
Leu Leu Lys Thr Arg Leu 755 76032229DNAArtificial SequenceDNA of
HSA-hIL-3 3atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta
ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga
tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt
atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta
actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga
caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc
ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct
360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc
ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca
atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat
ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc
tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc
caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag
660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc
atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag
aagtttccaa gttagtgaca 780gatcttacca aagtccacac ggaatgctgc
catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta
tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg
aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat
960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa
ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt
ttttgtatga atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg
ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc
tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc
ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag
1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa
gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc
taggaaaagt gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg
ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt
gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat
ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca
1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat
atgcacactt 1620tctgagaagg agagacaaat caagaaacaa actgcacttg
ttgagcttgt gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct
gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga
taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc
aagctgcctt aggcttagct cccatgaccc agacaacgtc cttgaagaca
1860agctgggtta actgctctaa catgatcgat gaaattataa cacacttaaa
gcagccacct 1920ttgcctttgc tggacttcaa caacctcaat ggggaagacc
aagacattct gatggaaaat 1980aaccttcgaa ggccaaacct ggaggcattc
aacagggctg tcaagagttt acagaacgca 2040tcagcaattg agagcattct
taaaaatctc ctgccatgtc tgcccctggc cacggccgca 2100cccacgcgac
atccaatcca tatcaaggac ggtgactgga atgaattccg gaggaaactg
2160acgttctatc tgaaaaccct tgagaatgcg caggctcaac agacgacttt
gagcctcgcg 2220atcttttag 22294718PRTArtificial SequenceHSA-hIL-3
4Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5
10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys
Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
Ala Pro Met Thr Gln Thr Thr 580 585 590Ser Leu Lys Thr Ser Trp Val
Asn Cys Ser Asn Met Ile Asp Glu Ile 595 600 605Ile Thr His Leu Lys
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn 610 615 620Leu Asn Gly
Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg625 630 635
640Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala
645 650 655Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu
Pro Leu 660 665 670Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile
Lys Asp Gly Asp 675 680 685Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe
Tyr Leu Lys Thr Leu Glu 690 695 700Asn Ala Gln Ala Gln Gln Thr Thr
Leu Ser Leu Ala Ile Phe705 710 71552313DNAArtificial SequenceDNA of
HSA-hEPO 5atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta
ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga
tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt
atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta
actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga
caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc
ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct
360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc
ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca
atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat
ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc
tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc
caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag
660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc
atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag
aagtttccaa gttagtgaca 780gatcttacca aagtccacac ggaatgctgc
catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta
tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg
aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat
960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa
ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt
ttttgtatga atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg
ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc
tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc
ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag
1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa
gaaagtaccc
1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt
gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag
aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa
acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa
caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca
aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt
1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt
gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg
atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc
tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt
aggcttaatc tgtgacagcc gagtcctgga gaggtacctc 1860ttggaggcca
aggaggccga gaatatcacg acgggctgtg ctgaacactg cagcttgaat
1920gagaatatca ctgtcccaga caccaaagtt aatttctatg cctggaagag
gatggaggtc 1980gggcagcagg ccgtagaagt ctggcagggc ctggccctgc
tgtcggaagc tgtcctgcgg 2040ggccaggccc tgttggtcaa ctcttcccag
ccgtgggagc ccctgcagct gcatgtggat 2100aaagccgtca gtggccttcg
cagcctcacc actctgcttc gggctctgcg agcccagaag 2160gaagccatct
cccctccaga tgcggcctca gctgctccac tccgaacaat cactgctgac
2220actttccgca aactcttccg agtctactcc aatttcctcc ggggaaagct
gaagctgtac 2280acaggggagg cctgcaggac aggggacaga tga
23136746PRTArtificial SequenceHSA-hEPO 6Asp Ala His Lys Ser Glu Val
Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu
Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr
Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His
Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg
Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90
95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala
Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu
Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys
Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys
Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg
Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215
220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr
Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu
Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu
Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu
Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu
Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp
Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu
Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330
335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu
Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu
Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Glu Val Ser Thr Pro
Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser
Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455
460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys
His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met
Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu Ile Cys Asp Ser Arg Val Leu
580 585 590Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr
Thr Gly 595 600 605Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr
Val Pro Asp Thr 610 615 620Lys Val Asn Phe Tyr Ala Trp Lys Arg Met
Glu Val Gly Gln Gln Ala625 630 635 640Val Glu Val Trp Gln Gly Leu
Ala Leu Leu Ser Glu Ala Val Leu Arg 645 650 655Gly Gln Ala Leu Leu
Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln 660 665 670Leu His Val
Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu 675 680 685Leu
Arg Ala Leu Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala 690 695
700Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg
Lys705 710 715 720Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys
Leu Lys Leu Tyr 725 730 735Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg
740 74572352DNAArtificial SequenceDNA of HSA-GCSF 7atgaagtggg
taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc
gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa
120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca
gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg
caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt
catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac
ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg
aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg
420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac
atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt
atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca
gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga
tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt
gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta
720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa
gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc
tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa
aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct
gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg
ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc
1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga
atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg
ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct
catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga
gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag
agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc
1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt
gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag
aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa
acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa
caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca
aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt
1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt
gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg
atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc
tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt
aggcttaacc cccctgggcc ctgccagctc cctgccccag 1860agcttcctgc
tcaagtgctt agagcaagtg aggaagatcc agggcgatgg cgcagcgctc
1920caggagaagc tgtgtgccac ctacaagctg tgccaccccg aggagctggt
gctgctcgga 1980cactctctgg gcatcccctg ggctcccctg agcagctgcc
ccagccaggc cctgcagctg 2040gcaggctgct tgagccaact ccatagcggc
cttttcctct accaggggct cctgcaggcc 2100ctggaaggga tctcccccga
gttgggtccc accttggaca cactgcagct ggacgtcgcc 2160gactttgcca
ccaccatctg gcagcagatg gaagaactgg gaatggcccc tgccctgcag
2220cccacccagg gtgccatgcc ggccttcgcc tctgctttcc agcgccgggc
aggaggggtc 2280ctagttgcct cccatctgca gagcttcctg gaggtgtcgt
accgcgttct acgccacctt 2340gcccagccct ga 23528759PRTArtificial
SequenceHSA-GCSF 8Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys
Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu
Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu
Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp
Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu
Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys
Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu
Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp
Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala
Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp
Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe
Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val
His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp
Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375
380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro 405 410 415Glu Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu Thr Pro Leu Gly Pro Ala Ser 580 585 590Ser Leu Pro
Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys 595 600 605Ile
Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr 610 615
620Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
Gly625 630 635 640Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln
Ala Leu Gln Leu 645 650 655Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly 660 665 670Leu Leu Gln Ala Leu Glu Gly Ile
Ser Pro Glu Leu Gly Pro Thr Leu 675 680 685Asp Thr Leu Gln Leu Asp
Val Ala Asp Phe Ala Thr Thr Ile Trp Gln 690 695 700Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly705 710 715 720Ala
Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val 725 730
735Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val
740 745 750Leu Arg His Leu Ala Gln Pro 75592211DNAArtificial
SequenceDNA of HSA-GMCSF 9atgaagtggg taacctttat ttcccttctt
tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag
gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt
gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg
taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat
240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa
attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact
gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa
gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat
gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat
atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc
540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga
taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga
aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt
ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt
tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca
aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac
840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag
taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca
ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct
gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa
ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg
attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact
1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt
gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac
aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg
ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct
tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac
atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc
1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt
caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag
ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca
ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat
caagaaacaa actgcacttg ttgagcttgt gaaacacaag 1680cccaaggcaa
caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag
1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa
aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttagca cccgcccgct
cgcccagccc cagcacgcag 1860ccctgggagc atgtgaatgc catccaggag
gcccggcgtc tcctgaacct gagtagagac 1920actgctgctg agatgaatga
aacagtagaa gtcatctcag aaatgtttga cctccaggag 1980ccgacctgcc
tacagacccg cctggagctg tacaagcagg gcctgcgggg cagcctcacc
2040aagctcaagg gccccttgac catgatggcc agccactaca agcagcactg
ccctccaacc 2100ccggaaactt cctgtgcaac ccagattatc acctttgaaa
gtttcaaaga gaacctgaag 2160gactttctgc ttgtcatccc ctttgactgc
tgggagccag tccaggagtg a 221110712PRTArtificial SequenceHSA-GMCSF
10Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp
Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe
Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala
Pro Ala Arg Ser Pro Ser 580 585 590Pro Ser Thr Gln Pro Trp Glu His
Val Asn Ala Ile Gln Glu Ala Arg 595 600 605Arg Leu Leu Asn Leu Ser
Arg Asp Thr Ala Ala Glu Met Asn Glu Thr 610 615 620Val Glu Val Ile
Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu625 630 635 640Gln
Thr Arg Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr 645 650
655Lys Leu Lys Gly Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln His
660 665 670Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gln Ile Ile
Thr Phe 675 680 685Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe Leu Leu
Val Ile Pro Phe 690 695 700Asp Cys Trp Glu Pro Val Gln Glu705
710111830DNAHomo sapiens 11atgaagtggg taacctttat ttcccttctt
tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag
gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt
gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg
taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat
240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa
attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact
gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa
gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat
gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat
atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc
540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga
taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga
aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt
ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt
tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca
aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac
840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag
taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca
ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct
gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa
ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg
attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact
1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt
gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac
aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg
ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct
tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac
atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc
1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt
caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag
ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca
ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat
caagaaacaa actgcacttg ttgagcttgt gaaacacaag 1680cccaaggcaa
caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag
1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa
aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttataa 183012609PRTHomo
sapiens 12Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser
Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser
Glu Val Ala 20 25 30His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys
Ala Leu Val Leu 35 40 45Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro
Phe Glu Asp His Val 50 55 60Lys Leu Val Asn Glu Val Thr Glu Phe Ala
Lys Thr Cys Val Ala Asp65 70 75 80Glu Ser Ala Glu Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu Cys Thr Val Ala Thr
Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110Asp Cys Cys Ala Lys
Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125His Lys Asp
Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140Asp
Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys145 150
155 160Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
Pro 165 170 175Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe
Thr Glu Cys 180 185 190Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu
Pro Lys Leu Asp Glu 195 200 205Leu Arg Asp Glu Gly Lys Ala Ser Ser
Ala Lys Gln Arg Leu Lys Cys 210 215 220Ala Ser Leu Gln Lys Phe Gly
Glu Arg Ala Phe Lys Ala Trp Ala Val225 230 235 240Ala Arg Leu Ser
Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255Lys Leu
Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265
270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile
275 280 285Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys
Cys Glu 290 295 300Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu
Val Glu Asn Asp305 310 315 320Glu Met Pro Ala Asp Leu Pro Ser Leu
Ala Ala Asp Phe Val Glu Ser 325 330 335Lys Asp Val Cys Lys Asn Tyr
Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350Met Phe Leu Tyr Glu
Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365Leu Leu Leu
Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380Cys
Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu385 390
395 400Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn
Cys 405 410 415Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn
Ala Leu Leu 420 425 430Val Arg Tyr Thr Lys Lys Val Pro Glu Val Ser
Thr Pro Thr Leu Val 435 440 445Glu Val Ser Arg Asn Leu Gly Lys Val
Gly Ser Lys Cys Cys Lys His 450 455 460Pro Glu Ala Lys Arg Met Pro
Cys Ala Glu Asp Tyr Leu Ser Val Val465 470 475 480Leu Asn Gln Leu
Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495Val Thr
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505
510Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala
515 520 525Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu
Lys Glu 530 535 540Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu
Val Lys His Lys545 550 555 560Pro Lys Ala Thr Lys Glu Gln Leu Lys
Ala Val Met Asp Asp Phe Ala 565 570 575Ala Phe Val Glu Lys Cys Cys
Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590Ala Glu Glu Gly Lys
Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605Leu
13600DNAHomo sapiens 13atgaactgtg tttgccgcct ggtcctggtc gtgctgagcc
tgtggccaga tacagctgtc 60gcccctgggc caccacctgg cccccctcga gtttccccag
accctcgggc cgagctggac 120agcaccgtgc tcctgacccg ctctctcctg
gcggacacgc ggcagctggc tgcacagctg 180agggacaaat tcccagctga
cggggaccac aacctggatt ccctgcccac cctggccatg 240agtgcggggg
cactgggagc tctacagctc ccaggtgtgc tgacaaggct gcgagcggac
300ctactgtcct acctgcggca cgtgcagtgg ctgcgccggg caggtggctc
ttccctgaag 360accctggagc ccgagctggg caccctgcag gcccgactgg
accggctgct gcgccggctg 420cagctcctga tgtcccgcct ggccctgccc
cagccacccc cggacccgcc ggcgcccccg 480ctggcgcccc cctcctcagc
ctgggggggc atcagggccg cccacgccat cctggggggg 540ctgcacctga
cacttgactg ggccgtgagg ggactgctgc tgctgaagac tcggctgtga
60014199PRTHomo sapiens 14Met Asn Cys Val Cys Arg Leu Val Leu Val
Val Leu Ser Leu Trp Pro1 5 10 15Asp Thr Ala Val Ala Pro Gly Pro Pro
Pro Gly Pro Pro Arg Val Ser 20 25 30Pro Asp Pro Arg Ala Glu Leu Asp
Ser Thr Val Leu Leu Thr Arg Ser 35 40 45Leu Leu Ala Asp Thr Arg Gln
Leu Ala Ala Gln Leu Arg Asp Lys Phe 50 55 60Pro Ala Asp Gly Asp His
Asn Leu Asp Ser Leu Pro Thr Leu Ala Met65 70 75 80Ser Ala Gly Ala
Leu Gly Ala Leu Gln Leu Pro Gly Val Leu Thr Arg 85 90 95Leu Arg Ala
Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp Leu Arg 100 105 110Arg
Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Thr 115 120
125Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met
130 135 140Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala
Pro Pro145 150 155 160Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile
Arg Ala Ala His Ala 165 170 175Ile Leu Gly Gly Leu His Leu Thr Leu
Asp Trp Ala Val Arg Gly Leu 180 185 190Leu Leu Leu Lys Thr Arg Leu
19515582DNAHomo sapiens 15atgggggtgc acgaatgtcc tgcctggctg
tggcttctcc tgtccctgct gtcgctccct 60ctgggcctcc cagtcctggg cgccccacca
cgcctcatct gtgacagccg agtcctggag 120aggtacctct tggaggccaa
ggaggccgag aatatcacga cgggctgtgc tgaacactgc 180agcttgaatg
agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg
240atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct
gtcggaagct 300gtcctgcggg gccaggccct gttggtcaac tcttcccagc
cgtgggagcc cctgcagctg 360catgtggata aagccgtcag tggccttcgc
agcctcacca ctctgcttcg ggctctgcga 420gcccagaagg aagccatctc
ccctccagat gcggcctcag ctgctccact ccgaacaatc 480actgctgaca
ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg
540aagctgtaca caggggaggc ctgcaggaca ggggacagat ga 58216193PRTHomo
sapiens 16Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
Ser Leu1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro
Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu
Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His
Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp Thr Lys Val Asn
Phe Tyr Ala Trp Lys Arg65 70 75 80Met Glu Val Gly Gln Gln Ala Val
Glu Val Trp Gln Gly Leu Ala Leu 85 90 95Leu Ser Glu Ala Val Leu Arg
Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110Gln Pro Trp Glu Pro
Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125Leu Arg Ser
Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu 130 135 140Ala
Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile145 150
155 160Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe
Leu 165 170 175Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg
Thr Gly Asp 180 185 190Arg17630DNAHomo sapiens 17ggatccatgg
ctggacctgc cacccagagc cccatgaagc tgatggccct gcagctgctg 60ctgtggcaca
gtgcactctg gacagtgcag gaagccaccc ccctgggccc tgccagctcc
120ctgccccaga gcttcctgct caagtgctta gagcaagtga ggaagatcca
gggcgatggc 180gcagcgctcc aggagaagct gtgtgccacc tacaagctgt
gccaccccga ggagctggtg 240ctgctcggac actctctggg catcccctgg
gctcccctga gcagctgccc cagccaggcc 300ctgcagctgg caggctgctt
gagccaactc catagcggcc ttttcctcta ccaggggctc 360ctgcaggccc
tggaagggat ctcccccgag ttgggtccca ccttggacac actgcagctg
420gacgtcgccg actttgccac caccatctgg cagcagatgg aagaactggg
aatggcccct 480gccctgcagc ccacccaggg tgccatgccg gccttcgcct
ctgctttcca gcgccgggca 540ggaggggtcc tagttgcctc ccatctgcag
agcttcctgg aggtgtcgta ccgcgttcta 600cgccaccttg cccagccctg
agccgaattc 63018204PRTHomo sapiens 18Met Ala Gly Pro Ala Thr Gln
Ser Pro Met Lys Leu Met Ala Leu Gln1 5 10 15Leu Leu Leu Trp His Ser
Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 20 25 30Leu Gly Pro Ala Ser
Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60Leu Cys Ala
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu65 70 75 80Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90
95Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
100 105 110Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu 115 120 125Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
Ala Asp Phe Ala 130 135 140Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
Gly
Met Ala Pro Ala Leu145 150 155 160Gln Pro Thr Gln Gly Ala Met Pro
Ala Phe Ala Ser Ala Phe Gln Arg 165 170 175Arg Ala Gly Gly Val Leu
Val Ala Ser His Leu Gln Ser Phe Leu Glu 180 185 190Val Ser Tyr Arg
Val Leu Arg His Leu Ala Gln Pro 195 20019448DNAHomo sapiens
19atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc
60cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg
120cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt
agaagtcatc 180tcagaaatgt ttgacctcca ggagccgacc tgcctacaga
cccgcctgga gctgtacaag 240cagggcctgc ggggcagcct caccaagctc
aagggcccct tgaccatgat ggccagccac 300tacaagcagc actgccctcc
aaccccggaa acttcctgtg caacccagat tatcaccttt 360gaaagtttca
aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag
420ccagtccagg agtgagaccg gccagatg 44820144PRTHomo sapiens 20Met Trp
Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile1 5 10 15Ser
Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25
30Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp
35 40 45Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met
Phe 50 55 60Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu
Tyr Lys65 70 75 80Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly
Pro Leu Thr Met 85 90 95Met Ala Ser His Tyr Lys Gln His Cys Pro Pro
Thr Pro Glu Thr Ser 100 105 110Cys Ala Thr Gln Ile Ile Thr Phe Glu
Ser Phe Lys Glu Asn Leu Lys 115 120 125Asp Phe Leu Leu Val Ile Pro
Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 14021459DNAHomo sapiens
21atgagccgcc tgcccgtcct gctcctgctc caactcctgg tccgccccgg actccaagct
60cccatgaccc agacaacgtc cttgaagaca agctgggtta actgctctaa catgatcgat
120gaaattataa cacacttaaa gcagccacct ttgcctttgc tggacttcaa
caacctcaat 180ggggaagacc aagacattct gatggaaaat aaccttcgaa
ggccaaacct ggaggcattc 240aacagggctg tcaagagttt acagaacgca
tcagcaattg agagcattct taaaaatctc 300ctgccatgtc tgcccctggc
cacggccgca cccacgcgac atccaatcca tatcaaggac 360ggtgactgga
atgaattccg gaggaaactg acgttctatc tgaaaaccct tgagaatgcg
420caggctcaac agacgacttt gagcctcgcg atcttttag 45922152PRTHomo
sapiens 22Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val
Arg Pro1 5 10 15Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Ser Leu Lys
Thr Ser Trp 20 25 30Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr
His Leu Lys Gln 35 40 45Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu
Asn Gly Glu Asp Gln 50 55 60Asp Ile Leu Met Glu Asn Asn Leu Arg Arg
Pro Asn Leu Glu Ala Phe65 70 75 80Asn Arg Ala Val Lys Ser Leu Gln
Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95Leu Lys Asn Leu Leu Pro Cys
Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110Arg His Pro Ile His
Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125Lys Leu Thr
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140Thr
Thr Leu Ser Leu Ala Ile Phe145 1502330DNAArtificial SequenceCloning
primer 23gaattcatga agtgggtaac ctttatttcc 302433DNAArtificial
SequenceCloning primer 24gaattcttat aagcctaagg cagcttgact tgc
332528DNAArtificial SequenceCloning primer 25catatgaact gtgtttgccg
cctggtcc 282625DNAArtificial SequenceCloning primer 26gatatgtatg
acacatttaa ttccc 252726DNAArtificial SequenceCloning primer
27ggatccatgg gggtgcacga atgtcc 262826DNAArtificial SequenceCloning
primer 28gaattctcat ctgtcccctg tcctgc 262925DNAArtificial
SequenceCloning primer 29ggatccatgg ctggacctgc caccc
253026DNAArtificial SequenceCloning primer 30gaattctcag ggctgggcaa
ggtggc 263128DNAArtificial SequenceCloning primer 31ggatccatgt
ggctgcagag cctgctgc 283225DNAArtificial SequenceCloning primer
32gaattctcac tcctggactg gctcc 253333DNAArtificial SequenceCloning
primer 33ctgccttagg cttacctggg ccaccacctg gcc 333429DNAArtificial
SequenceCloning primer 34tgtcgactca cagccgagtc ttcagcagc
293533DNAArtificial SequenceCloning primer 35ctgccttagg cttaatctgt
gacagccgag tcc 333628DNAArtificial SequenceCloning primer
36cactcgagtc atctgtcccc tgtcctgc 283735DNAArtificial
SequenceCloning primer 37ctgccttagg cttaaccccc ctgggccctg ccagc
353825DNAArtificial SequenceCloning primer 38ctcgagtcag ggctgggcaa
ggtgg 253935DNAArtificial SequenceCloning primer 39actccttagg
cttagcaccc gcccgctcgc ccagc 354025DNAArtificial SequenceCloning
primer 40ctcgagtcac tcctggactg gctcc 25
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