U.S. patent number 10,385,376 [Application Number 15/523,107] was granted by the patent office on 2019-08-20 for 21-hydroxylation of steroids.
This patent grant is currently assigned to SANOFI. The grantee listed for this patent is SANOFI. Invention is credited to Simone Anderko, Rita Bernhardt, Frank Hannemann, Bernd Janocha, Claus Lattemann, Hans-Falk Rasser, Sebastian Rissom, Thomas Stillger.
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United States Patent |
10,385,376 |
Lattemann , et al. |
August 20, 2019 |
21-hydroxylation of steroids
Abstract
Generally, the present invention relates to the field of steroid
hydroxylation. More specifically, the present invention relates to
a method for the 21-hydroxylation of steroids in cells. It also
relates to cells expressing a steroid 21-hydroxylating enzyme or
steroid 21-hydroxylase, expression vectors comprising a nucleic
acid encoding for a steroid 21-hydroxylase and a kit for carrying
out the method for the 21-hydroxylation of steroids in cells.
Inventors: |
Lattemann; Claus (Frankfurt am
Main, DE), Stillger; Thomas (Frankfurt am Main,
DE), Janocha; Bernd (Frankfurt am Main,
DE), Rasser; Hans-Falk (Frankfurt am Main,
DE), Rissom; Sebastian (Frankfurt am Main,
DE), Anderko; Simone (Saarbrucken, DE),
Bernhardt; Rita (Saarbrucken, DE), Hannemann;
Frank (Saarbrucken, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
SANOFI |
Paris |
N/A |
FR |
|
|
Assignee: |
SANOFI (Paris,
FR)
|
Family
ID: |
51932297 |
Appl.
No.: |
15/523,107 |
Filed: |
October 29, 2015 |
PCT
Filed: |
October 29, 2015 |
PCT No.: |
PCT/EP2015/075096 |
371(c)(1),(2),(4) Date: |
April 28, 2017 |
PCT
Pub. No.: |
WO2016/066738 |
PCT
Pub. Date: |
May 06, 2016 |
Prior Publication Data
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|
|
|
Document
Identifier |
Publication Date |
|
US 20170321241 A1 |
Nov 9, 2017 |
|
Foreign Application Priority Data
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|
|
|
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Oct 30, 2014 [EP] |
|
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14306740 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P
33/06 (20130101) |
Current International
Class: |
C12P
33/06 (20060101) |
Other References
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.
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P450 3A4 by Co-Expression with Human Molecular Chaperone HDJ-1
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2004). cited by examiner .
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production of 20.beta.OH-NorDHCMT, a long-term metabolite of
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Yeast Co-Expression System" Human Mutation, 1044, E443-E450, 2008
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co-expression with human molecular chaperone HDJ-1(Hsp40)", Protein
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3389-3402, Oxford University Press. cited by applicant .
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fission yeast", Dissertation, University of Saarbrucken, 2010,
Saarbrucken, Germany, pp. i-xiv-1-100, XP002751430, Retrieved from
the Internet <URL:
http://scidok.sulb.uni-saarland.de/volltexte/2010/3485/pdf/phd_c-
ummulative_dragan_upload.pdf>. cited by applicant .
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64, 2012, pp. 506-512, Wiley. cited by applicant .
Gupta et al: "Co-expression of chaperonin GroEL/GroES enhances in
vivo folding of yeast mitochondrial aconitase and alters the growth
characteristics of Escherichia coli", The International Journal of
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cell mediated steroid synthesis and molecular evaluation of steroid
hydroxylases", Journal of Biotechnology, vol. 124, 2006, pp.
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CYP105A1-based whole-cell biocatalyst for the conversion of resin
acid diterpenoids in permeabilized Escherichia coli", Applied
Microbiology and Biotechnology, vol. 37, Jun. 23, 2013 (Jun. 23,
2013), pp. 7639-7649, Springer. cited by applicant .
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and export of organophosphorus hydrolase in Escherichia coli",
Biotechnology Progress, vol. 15, 2012, pp. 925-930, Wiley. cited by
applicant .
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high-scoring segments in molecular sequences." Proc. Natl. Acad.
Sci. USA 1993 90:5873-5877, National Academies Press. cited by
applicant .
Lah et al: "The versatility of the fungal cytochrome P450
monooxygenase system is instrumental in xenobiotic detoxification",
Molecular Microbiology, vol. 81, 2011, pp. 1374-1389,
Wiley-Blackwell. cited by applicant .
Ringle et al: "Application of a new versatile electron transfer
system for cytochrome P450-based Escherichia coli whole-cell
bioconversions", Applied Microbiology and Biotechnology, vol. 97,
Dec. 20, 2012 (Dec. 20, 2012), pp. 7741-7754, Springer. cited by
applicant .
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microsomal cytochrome P450s CYP17 and CYP21 involved in steroid
hormone biosynthesis", Biochemistry (Moscow), vol. 77, 2012, pp.
585-592, Springer. cited by applicant .
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perspectives for synthetic application", Trends in Biotechnology,
vol. 30, 2012, pp. 26-36, Elsevier. cited by applicant .
Yoshimoto et al: "Minor activities and transition state properties
of the human steroid hydroxylases cytochromes P450c17 and P450c21,
from reactions observed with deuterium-labeled substrates",
Biochemistry, vol. 51, 2012, pp. 7064-7077, American Chemical
Society. cited by applicant .
Zehentgruber et al: "Challenges of steroid biotransformation with
human cytochrome P450 monooxygenase CYP21 using resting cells of
recombinant Schizosaccharomyces pompe", Journal of Biotechnology,
vol. 146, 2010, pp. 179-185, Elsevier. cited by applicant .
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a competitive inhibitor of human cytochromes P450c17 and P450c21",
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by applicant .
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applicant.
|
Primary Examiner: Berke-Schlessel; David W
Attorney, Agent or Firm: McDonnell Boehnen Hulbert &
Berghoff LLP
Claims
The invention claimed is:
1. A process for the hydroxylation of the carbon atom 21 of a
steroid, comprising the steps of: (a) providing a cell expressing:
(i) a heterologous CYP21A2 protein or a functional variant thereof;
(ii) at least one heterologous electron transfer system capable of
transferring electrons to CYP21A2; and (iii) one or more chaperones
facilitating folding of CYP21A2; and (b) adding the steroid to the
cell, wherein the steroid is medrane or deltamedrane.
2. The process of claim 1, further comprising a step (c) of
extracting the 21-hydroxylated steroid from the supernatant of the
cell.
3. The process of claim 1, further comprising adding one or more
cell permeabilizing agents to the cell after step (b).
4. The process of claim 1, wherein the cell is a resting cell.
5. The process of claim 1, wherein the cell is a prokaryotic cell
or a eukaryotic cell.
6. The process of claim 1, wherein the at least one heterologous
electron transfer system comprises: (a) a CYP21A2 reductase, and/or
(b) a ferredoxin reductase.
7. The process of claim 1, wherein the one or more chaperones are
recombinantly expressed chaperones.
8. The process of claim 1, wherein the expression of at least one
tryptophanase gene is reduced or abolished in the cell.
9. The process of claim 1, wherein the cell further expresses a
heterologous gene encoding for an enzyme catalyzing a step in the
heme biosynthesis pathway.
10. The process of claim 1, wherein the genes encoding for (i),
(ii), and optionally (iii) are comprised in one or more expression
cassettes which are integrated into the cell genome.
11. The process of claim 5, wherein the cell is an E. coli
cell.
12. The process of claim 5, wherein the cell is a yeast cell.
13. The process of claim 9, wherein the heterologous gene encoding
for an enzyme catalyzing a step in the heme biosynthesis pathway is
a hemA gene.
14. The process of claim 6, wherein the at least one heterologous
electron transfer system comprises an NADPH-dependent ferredoxin
reductase and a ferredoxin.
Description
This application is a national stage application under 35 U.S.C.
.sctn. 371 of International Application No. PCT/EP2015/075096,
filed Oct. 29, 2015, which claims the benefit of European
Application No. EP 14306740.3, filed Oct. 30, 2014, the disclosures
of which are explicitly incorporated herein in their entirety by
reference.
Generally, the present invention relates to the field of steroid
hydroxylation. More specifically, the present invention relates to
a method for the 21-hydroxylation of steroids in cells. It also
relates to cells expressing a steroid 21-hydroxylating enzyme or
steroid 21-hydroxylase, expression vectors comprising a nucleic
acid encoding for a steroid 21-hydroxylase and a kit for carrying
out the method for the 21-hydroxylation of steroids in cells.
Synthetic glucocorticoids are descendent from the natural occurring
stress hormone cortisol and play a crucial role in pharmaceutical
industry because of their anti-inflammatory and immune suppressive
effects. Moreover, synthetic molecules often act more effective
than cortisol.
Currently, the synthesis of some pharmaceutically active steroids
involves a 21-hydroxylation of their precursor (see FIG. 1 for an
example), which consists of a long lasting chemical multistep
synthesis. By means of synthetic chemistry this hydroxylation is
also difficult, as the chemical oxidants are not selective to
position 21. For this reason, other functional groups have to be
protected to avoid their oxidation and to direct the hydroxylation
reaction towards position 21. Furthermore, the synthesis is not
environmentally friendly because of the use of reagents such as
iodine. Therefore, a cheap and sustainable production of
pharmaceutically active steroids is highly desirable to satisfy the
high demand for these important drugs.
This problem has been addressed by the present inventors by the
development of whole cell biotransformation of steroids in a
one-step synthesis by the enzyme CYP21A2, which is a member of the
protein family of the cytochrome P450 monooxygenases and which is
able to perform a highly selective hydroxylation of steroids at the
21-position of the steroid scaffold (see FIG. 2 for a scheme of the
process). CYP21A2 is a mammalian membrane anchored enzyme which is
located in the endoplasmic reticulum and which plays a crucial role
in the steroid hormone biosynthesis. The inventors have shown that
the biocatalytic system of the invention is a promising candidate
to replace the established chemical synthesis. In particular, they
have shown that steroids could be modified within one single
hydroxylation step, leading to the one desired product, which is
saving time, is environmentally friendly (no by-products were
observed) and facilitates downstream processing. Furthermore and
advantageously, for the steroid production in whole cells according
to the invention, enzymes do not have to be purified, remain stable
in the host and the addition of costly redox equivalents like NADPH
is not necessary, because the cell itself serves as a donor.
Before the present invention is described in detail below, it is to
be understood that this invention is not limited to the particular
methodology, protocols and reagents described herein as these may
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention which
will be limited only by the appended claims. Unless defined
otherwise, all technical and scientific terms used herein have the
same meanings as commonly understood by one of ordinary skill in
the art.
Preferably, the terms used herein are defined as described in "A
multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kolbl, H.
eds. (1995), Helvetica Chimica Acta, CH-4010 Basel,
Switzerland).
Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions etc.), whether supra or
infra, is hereby incorporated by reference in its entirety. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
In the following, the elements of the present invention will be
described. These elements are listed with specific embodiments,
however, it should be understood that they may be combined in any
manner and in any number to create additional embodiments. The
variously described examples and preferred embodiments should not
be construed to limit the present invention to only the explicitly
described embodiments. This description should be understood to
support and encompass embodiments which combine the explicitly
described embodiments with any number of the disclosed and/or
preferred elements. Furthermore, any permutations and combinations
of all described elements in this application should be considered
disclosed by the description of the present application unless the
context indicates otherwise.
Throughout this specification and the claims which follow, unless
the context requires otherwise, the word "comprise", and variations
such as "comprises" and "comprising", are to be understood to imply
the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group
of integer or step. As used in this specification and the appended
claims, the singular forms "a", "an", and "the" include plural
referents, unless the content clearly dictates otherwise.
In a first aspect, the present invention relates to a process for
the hydroxylation of the carbon atom 21 of a steroid, comprising
the steps of:
a) providing a cell expressing
(i) a heterologous CYP21A2 protein or a functional variant thereof,
(ii) at least one heterologous electron transfer system capable of
transferring electrons to CYP21A2, and (iii) one or more chaperones
facilitating folding of CYP21A2; and b) adding the steroid to the
cell.
A steroid is a type of organic compound that contains a
characteristic arrangement of four cycloalkane rings that are
joined to each other (shown below). The core of steroids is
composed of seventeen carbon atoms bonded together that take the
form of four fused rings: three cyclohexane rings (designated as
rings A, B and C) and one cyclopentane ring (the D ring). The
steroids vary by the functional groups attached to this four-ring
core and by the oxidation state of the rings.
The hydroxylation of the carbon atom 21 of a steroid is the
addition of an --OH group at position 21 as shown in the
above-shown ABCD steroid ring system. The numbering of the carbon
atoms is according to the IUPAC (International Union of Pure and
Applied Chemistry)-approved ring lettering and atom numbering. The
21-hydroxylation of a steroid is shown in FIG. 1.
In a particular embodiment of the process of the first aspect of
the invention, the steroid is a 3-keto steroid. More particularly,
the steroid is a non-natural steroid, i.e. a steroid that is not
produced and/or 21-hydroxylated in cells, especially human or
bovine cells, which are not genetically altered.
In one embodiment, the steroid is selected from the group
consisting of medrane, deltamedrane, progesterone,
17OH-progesterone, medroxyprogesterone, and
5-.alpha.-dihydro-progesterone. The 21-hydroxylation converts these
particular steroids to premedrol, medrol, 11-deoxycorticosterone,
11-deoxycortisol, 21OH-medroxyprogesterone and
21OH-(5.alpha.-dihydroprogesterone), respectively. In one
particular embodiment, in which the steroid is a non-natural
steroid, the steroid is selected from the group consisting of
medrane, deltamedrane, medroxyprogesterone, and
5-.alpha.-dihydro-progesterone.
The cell is in particular a cultured cell, cultured in any cell
medium, e.g. in a growth medium, and is, in a particular
embodiment, in a resting state. According to this embodiment, the
cell is comprised in a buffer or medium capable of maintaining the
cell rather than in a growth medium. The composition of the buffer
depends on the particular cell and suitable buffers are well-known
in the art. Depending on the cell-type, the cell is a suspension
cell or an adherent cell. A suspension cell is a cell that may
naturally live in suspension (i.e. without being attached to a
surface), or a cell that has been modified to be able to survive in
suspension cultures, for example to be grown to higher densities
than adherent conditions would allow. An adherent cell is a cell
that requires a surface, such as tissue culture plastic or
microcarrier, which may be coated with extracellular matrix (such
as collagen and laminin) components to increase adhesion properties
and provide other signals needed for growth and differentiation. In
one embodiment, the adherent cell is a monolayer cell.
Generally, the cell may be a prokaryotic cell or a eukaryotic cell.
A particular example of a prokarytic cell to be used in the process
of the first aspect is an E. coli cell, e.g. of the E. coli strain
C43(DE3) of the examples. A particular example of a eukaryotic cell
is a yeast cell, e.g. a S. cerevisae cell or a Schizosaccharomyces
pombe cell. However, the process of the first aspect is not limited
to any particular cell type and any cell type may be used, in
particular any cell type that can be grown and maintained in
culture and that can be used as a recombinant expression system,
like insect cells or mammalian cells in addition to the
above-mentioned cells.
The term "heterologous" means that a protein is expressed in cell
that does not normally (i.e. without human intervention) express
that protein. CYP21A2, for example is an enzyme also called
21-hydroxylase, which is part of the cytochrome P450 family of
enzymes. Cytochrome P450 enzymes are involved in many processes in
the body, such as assisting with reactions that break down drugs
and helping to produce cholesterol, certain hormones, and fats
(lipids). The 21-hydroxylase enzyme is found in the adrenal glands,
which are located on top of the kidneys and produce a variety of
hormones that regulate many essential functions in the body.
Therefore, with respect to heterologous expression, the CYP21A2
protein can be considered heterologous regarding any cell which is
not an adrenal gland cell.
In particular, the term "heterologous" can also refer to the
species a protein or gene is derived from in comparison to the cell
in which it is expressed, in particular recombinantly expressed. A
heterologous protein is then a protein that is derived from a
different species than the cell it is expressed in, i.e. the cell
of the process of the first aspect of the invention.
For example, the present inventors expressed mammalian, in
particular human or bovine proteins in an E. coli cell, making
these proteins heterologous with respect to the cell.
In a particular embodiment of the process of the first aspect of
the invention, the CYP21A2 protein is of human or bovine origin.
Human CYP21A2 (UniProt accession number P08686) has the sequence
according to SEQ ID NO: 1 of the sequence listing. Bovine CYP21A2
(UniProt accession number P00191) has the sequence according to SEQ
ID NO: 2 of the sequence listing. See also FIG. 8. Homologous
genes, however, do also exist in other mammalian species, such as
Canis lupus (dog), Macaca mulata (resus monkey), Rattus norvegicus
(rat), Gallus gallus (chicken), Danio rerio (zebra fish), Mus
musculus (mouse), or Pan troglodytes (chimpanzee). Therefore, it is
emphasized that any CYP21A2 protein can be used, in particular any
mammalian CYP21A2 protein. A modified human or bovine CYP21A2
according to SEQ ID NO: 3 and SEQ ID NO: 4, respectively, can also
be used. See also FIG. 8.
The term "functional variant" is a protein variant that has at
least 20% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%;
95%; 98%; 99%; 99.5%; or 100% or even more) of the ability of the
unaltered or CYP21A2 protein to 21-hydroxylate a steroid. This
ability can be determined by the skilled person without undue
burden using, for example, the methods shown in Examples 1 and 2
herein.
A "protein variant" is a protein that has an amino acid sequence
that it at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to the amino acid sequence of the CYP21A2 it is derived
from, for example SEQ ID NO: 1 or 3 in case of human CYP21A2 or SEQ
ID NO: 2 or 4 in case of bovine CYP21A2. The determination of
percent identity between two sequences is accomplished using the
mathematical algorithm of Karlin and Altschul, Proc. Natl. Acad.
Sci. USA 90, 5873-5877, 1993. Such an algorithm is incorporated
into the BLASTN and BLASTP programs of Altschul et al. (1990) J.
Mol. Biol. 215, 403-410. To obtain gapped alignments for
comparative purposes, Gapped BLAST is utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs are used.
Alternatively, a protein variant can also be defined as having up
to 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino
acid substitutions, in particular conservative amino acid
substitutions. Conservative substitution tables are well known in
the art (see for example Creighton (1984) Proteins. W. H. Freeman
and Company). An overview of physical and chemical properties of
amino acids is given in Table 1 below. In a particular embodiment,
conservative substitutions are substitution made with amino acids
have the same properties according to Table 1.
TABLE-US-00001 TABLE 1 Properties of naturally occuring proteins.
Charge properties/ hydrophobicity Side group Amino Acid nonpolar
hydrophobic aliphatic Ala, Ile, Leu, Val aliphatic, S-containing
Met aromatic Phe, Trp imino Pro polar uncharged aliphatic Gly amide
Asn, Gln aromatic Tyr hydroxyl Ser, Thr sulfhydryl Cys positively
charged basic Arg, His, Lys negatively charged acidic Asp, Gly
The term "variant" also includes protein fragments. A fragment of
CYP21A2 has an N-terminal and/or C-terminal deletion of up to 100,
90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acids in
total. In a particular embodiment, the functional variant is a
fragment of CYP21A2 lacking the hydrophobic anchor region, i.e. a
truncation of 29 N-terminal amino acid residues (FIG. 8).
In addition, the CYP21A2 protein may be modified, for example by
N-terminal or C-terminal amino acid additions, such as tags or
N-terminal modifications for improved bacterial expression. For
example, the tag may be a C-terminal His-tag, e.g. 6.times. His-tag
and the N-terminal modification an addition of ten amino acids from
the N-terminus of CYP2C3 (see FIG. 8 and SEQ ID Nos 3 and 4).
An electron transfer system is a series of compounds that transfer
electrons from electron donors to electron acceptors via redox
reactions. The term "capable of transferring electrons to CYP21A2"
thereby means that CYP21A2 is the electron acceptor of this series.
The term "series" herein may include CYP21A2 and, thus, the
electron transfer system may consist of only one protein in
addition to CYP21A2. For the process of the first aspect of the
invention, it is not crucial which members the electron transfer
system consists of, as long as the system is capable of
transferring electrons to CYP21A2. Suitable systems are well-known
in the art. In a particular embodiment of the process of the first
aspect of the invention, the at least one electron transfer system
comprises (i) a CYP21A2 reductase, or (ii) a ferredoxin reductase,
preferably an NADPH-dependent ferredoxin reductase (adrenodoxin
reductase) and a ferredoxin. For example, the at least one electron
transfer protein can be selected from the group consisting of an
NADPH-dependent cytochrome P450 reductase (CPR, e.g. human or
bovine), the combination of an adrenodoxin reductase (AdR, e.g.
human or bovine) and an adrenodoxin (Adx4-108, e.g. human or
bovine), the combination of a flavodoxin reductase (Fpr, e.g. from
E. coli) and an adrenodoxin (Adx4-108, e.g. human or bovine), the
combination of an adrenodoxin reductase homolog (Arh), e.g. S.
pombe adrenodoxin reductase homolog (Arh1), and an adrenodoxin
(Adx4-108, e.g. human or bovine), the combination of an adrenodoxin
reductase homolog (Arh), e.g. S. pombe adrenodoxin reductase
homolog (Arh1), and the ferredoxin domain of an electron transfer
domain (etp.sup.fd), e.g. the ferredoxin domain of the S. pombe
electron transfer domain (etp1.sup.fd), and the combination of a
flavodoxin reductase (Fpr, e.g. from E. coli) and the ferredoxin
domain of an electron transfer domain (etp.sup.fd), e.g. the
ferredoxin domain of the S. pombe electron transfer domain
(etp1.sup.fd).
In a particular embodiment, exogenous NADPH is not added to the
cell. In this embodiment, NADPH is produced by the cell. In a
specific embodiment, the NADPH-production in the cell may be
increased by recombinant means, e.g. the heterologous expression of
enzymes involved in NADPH production (e.g. one or more of
glucose-6-phosphate dehydrogenase (G6PD), phosphogluconate
dehydrogenase (PGD), malate dehydrogenase (MDH), and/or isocitrate
dehydrogenase (ICDH)), and/or it may be increased by providing
hormonal signals, i.e. adding hormones such as estradiol that
enhance the level of endogenous NADPH production.
Similarly, the chaperone can be any chaperone as long as it is
capable of facilitating folding of CYP21A2. The present inventors
found that the process of the first aspect of the invention can
rely only on endogenous chaperones, i.e. additional expression of
suitable chaperones is not essential. However, an additional
expression improves the production of functional CYP21A2 in the
cell and can therefore be beneficial. Thus, in one embodiment of
the process of the first aspect of the invention, the one or more
chaperones are recombinantly expressed chaperones. In particular,
the one or more chaperones may be heterologous chaperones.
Exemplary chaperones are the E. coli chaperones GroEL and GroES or
other chaperones like DnaK, DnaJ, GrpE, and ClpB as well as small
heat shock proteins (sHSP) such as IbpA and IbpB (IbpAB).
Optionally, the same chaperone or one or more additional
chaperone(s) to be expressed in the cell is/are capable of folding
one or more of above electron transfer proteins.
Recombinant expression refers to the expression of a recombinant
gene. Such a gene can be any gene introduced into the cell by
methods of genetic engineering and is usually a heterologous gene
and/or a gene regulated differently than a possible endogenous
counterpart gene in terms of expression. In one embodiment, the
recombinant expression is inducible.
In one embodiment of the process of the first aspect of the
invention, the process further comprises adding one or more cell
permeabilizing agents to the suspension of cells, for example after
step b). Cell permeabilizing agents are reagents which increase the
permeability of membranes. Examples are organic solvents, such as
methanol, acetone or DMSO, detergents such as saponin, Triton X-100
or Tween-20, and EDTA. In particular, polymyxin B can be used as a
cell permeabilizing agent. This agent proved to work particularly
well in the process of the invention.
In another embodiment of the process of the first aspect of the
invention, the process further comprises a step c) of extracting
the 21-hydroxylated steroid from the cell and/or the supernatant of
the cell (i.e. the buffer or medium the cell is comprised in), for
example from a whole cell suspension. The extraction can be done
with any solvents or extraction methods known in the art for
extracting undissolved compounds. For example, a solvent such as
1-butanol, 2-butanone or chloroform may be used.
In a specific embodiment of the process of the first aspect of the
invention, the expression of at least one tryptophanase gene is
reduced or abolished in the cell. A tryptophanase or L-tryptophan
indole-lyase (EC number 4.1.99.1.) is an enzyme catalyzing the
reaction L-tryptophan+H2O=indole+pyruvate+NH3. Accordingly, the
reduction or abolishment will lead to the decrease of indol
production by the cell, which can improve the process, since indol
is an inhibitor of CYP enzymes such as CYP21A2. As tryptophanase
genes are generally (but not exclusively) found in prokaryotes,
e.g. E. coli, this specific embodiment applies in particular to the
embodiment of the process of the first aspect of the invention in
which the cell is a prokaryotic cell. In one embodiment, the
species of a cell expressing a tryptophansae gene is selected from
the group consisting of Aeromonas hydrophila, Bacillus sp.,
Bacteroides sp., Corynebacterium sp., Enterobacter aerogenes,
Enterobacter aerogenes SM-18, Enterobacter sp., Erwinia sp.,
Escherichia aurescens, Escherichia coli, Fusobacterium necrophorum
subsp. Funduliforme, Kluyvera sp., Micrococcus aerogenes,
Morganella morganii, Paenibacillus alvei, Paracolobactrum
coliforme, Paracolobactrum sp., Pasteurella sp., Photobacterium
profundum, Porphyromonas gingivalis, Prevotella intermedia, Proteus
vulgaris, Providencia rettgeri, Shigella alkalescens, Sphaerophorus
sp., Symbiobacterium thermophilum, Vibrio sp. and a mammalian
species such as Homo sapiens and Rattus norvegicus,
In a further embodiment of the process of the first aspect of the
invention, the cell further expresses a heterologous or recombinant
gene encoding for an enzyme catalyzing a step in the heme
biosynthesis pathway, in particular a heterologous hemA (glutamyl
tRNA reductase) gene. An example for such cells is E. coli. This
will advantageously reduce the need for feeding precursors for the
synthesis of the CYP heme, such as the heme precursor
.delta.-aminolevulinic acid, and reduce the costs for the
biotransformation of steroids in such cells.
In a particular embodiment of the process of the first aspect of
the invention, the nucleic acids encoding for (i) a heterologous
CYP21A2 protein or a functional variant thereof, (ii) at least one
heterologous electron transfer system capable of transferring
electrons to CYP21A2, and optionally (iii) one or more chaperones
facilitating folding of CYP21A2 are comprised in one or more
expression cassette(s) which is/are integrated into the cell, in
particular into its genome. The term "expression cassette" refers
to a DNA fragment which comprises a gene operably linked to a
regulatory sequence such as a promoter, necessary for gene
expression. "Operably linked" refers to the linking of nucleotide
regions encoding specific genetic information such that the
nucleotide regions are contiguous, the functionality of the region
is preserved and will perform relative to the other regions as part
of a functional unit. The nucleic acids (i), (ii) and optionally
(iii) may each be comprised in individual expression cassettes or
in one or more multicistronic expression cassettes. As used herein,
the term "multicistronic" means that multiple cistrons, namely,
multiple nucleic acids or genes, are operably linked to the same
regulatory sequence, e.g. a promoter.
In one embodiment, the nucleic acids encoding for (i) a
heterologous CYP21A2 protein or a functional variant thereof, (ii)
at least one heterologous electron transfer system capable of
transferring electrons to CYP21A2, and optionally (iii) one or more
chaperones facilitating folding of CYP21A2 are comprised in an
expression vector comprised in the cell. An "expression vector" is
a vehicle by means of which DNA fragments that contain nucleic
acids encoding for a protein can be introduced into host cells
where the nucleic acids can be expressed by the host cell. The
nucleic acids (i), (ii) and optionally (iii) may each be comprised
in individual expression vectors or in one or more multicistronic
expression vectors.
Also, one or more of the nucleic acids (i), (ii) and optionally
(iii) may be comprised in an expression cassette which is
integrated into the cell genome, whereas the remaining nucleic
acids (i), (ii) or optionally (iii) are comprised in an expression
expression vector, both as set out above.
In a second aspect, the present invention relates to cell
expressing
(i) a heterologous CYP21A2 protein or a functional variant
thereof,
(ii) at least one heterologous electron transfer system capable of
transferring electrons to CYP21A2, and
(iii) one or more chaperones facilitating folding of CYP21A2.
This cell as well as the cell of the process of the first aspect of
the invention can also be described as a cell comprising one or
more nucleic acids encoding for
(i) a heterologous CYP21A2 protein or a functional variant
thereof,
(ii) at least one heterologous electron transfer system capable of
transferring electrons to CYP21A2, and
(iii) one or more chaperones facilitating folding of CYP21A2.
In a particular embodiment, these nucleic acids are comprised in
one or more expression cassettes.
The cell of the second aspect is in essence the cell used in the
process of the first aspect of the invention and, therefore,
further embodiments of the cell of the second aspect of the
invention are as described above with respect to the process of the
first aspect.
In a third aspect, the present invention relates to a
multicistronic expression vector comprising (i) a nucleic acid
encoding for a CYP21A2 protein or a functional variant thereof,
(ii) one or more nucleic acids encoding for at least one
heterologous electron transfer system capable of transferring
electrons to CYP21A2, and optionally (iii) one or more nucleic
acids encoding for chaperones facilitating folding of CYP21A2.
In a fourth aspect, the present invention relates to a kit
comprising a cell of the second aspect, a multicistronic expression
vector of the third aspect, or (i) an expression vector comprising
a nucleic acid encoding for a CYP21A2 protein or a functional
variant thereof, (ii) one or more expression vectors comprising one
or more nucleic acids encoding for at least one heterologous
electron transfer system capable of transferring electrons to
CYP21A2, and optionally (iii) one or more expression vectors
comprising one or more nucleic acids encoding for chaperones
facilitating folding of CYP21A2.
The term "kit" is used herein to mean a collection of all or some
of the reagents, materials, and instructions necessary to carry out
the process of the first aspect. This includes cell culture medium
or buffer (both in dry or liquid form), one or more cell
permeabilizing agents, solvents for extracting the steroid and, in
particular, the steroid to be hydroxylated, in particular a 3-keto
steroid, for example one or more of medrane, deltamedrane,
progesterone, 17OH-progesterone, medroxyprogesterone, or
5.alpha.-dihydroprogesterone. Furthermore, it is envisaged that all
reagents or materials described herein with relation to the process
of the first aspect can be part of the kit of the fourth
aspect.
In a particular embodiment, the process of the first aspect, the
cell of the second aspect, the expression vector of the third
aspect and the kit of the fourth aspect do not comprise a further
step or component as applicable, especially a heterologous gene or
protein, for steroid conversion or production which is not related
to the 21-hydroxylation of steroids, particularly as described
herein. Optionally, an exception of this may be downstream steps or
components related to the further processing of the 21-hydroxylated
steroid (such as the conversion from premedrol to medrol) or
upstream steps or components related to the production of the
steroid to be 21-hydroxylated (such as the production of
medrane).
In the following figures and examples, some particular embodiments
of the invention are described in more detail. Yet, no limitation
of the invention is intended by the details of the particular
embodiments. In contrast, the invention pertains to any embodiment
which comprises details which are not explicitly mentioned in the
embodiments herein, but which the skilled person finds without
undue effort.
DESCRIPTION OF THE FIGURES
FIG. 1: Hydroxylation of a steroid (here progesterone) at carbon
atom 21.
FIG. 2: Scheme of a whole cell biotransformation of a steroid by
CYP21A2 and the needed electron transfer proteins in E. coli.
FIG. 3: left: CO difference spectrum of purified bovine CYP21;
right: SDS-PAGE of bCYP21 samples taken after indicated
purification steps (IMAC/DEAE/SP).
FIG. 4: HPLC chromatogram of the in vitro 21-hydroxylation of
medrane to premedrol by human CYP21 and described electron transfer
proteins, here AdR and Adx (system 2).
FIGS. 5A-5B: Constructed vectors for whole cell biotransformation
using human or bovine CYP21 with different electron transfer
proteins.
FIGS. 6A-D: HPLC chromatogram of the whole cell 21-hydroxylation of
medrane to premedrol (A), delta-medrane to medrol (B),
medroxyprogesterone to 21OH-medroxyprogesterone (C), and
17OH-progesterone to 11-deoxycortisol (D) by bovine CYP21 and
described electron transfer proteins, here Fpr and Adx.
FIG. 7: Time-dependent whole cell conversion of medrane to
premedrol by bovine CYP21 and described electron transfer proteins,
here Fpr and Adx.
FIGS. 8A-8B: Amino acid sequences of wildtype and modified human
(A) and bovine (B) CYP21A2.
DESCRIPTION OF THE EXAMPLES
Example 1: In Vitro Hydroxylation
1.1 Expression/Purification of hCYP21/bCYP21
To show that both human and bovine CYP21 are able to hydroxylate
steroids at position 21, in vitro studies with both enzymes were
performed. As an exemplary 21-hydroxylation process, medrane was
converted to premedrol:
##STR00001##
Premedrol (methylhydrocortisone) is a precursor of a highly
effective pharmaceutical steroid medrol (methylprednisolone).
Medrol is an important drug in therapy of autoimmune diseases,
multiple sclerosis and in general for local and systematic
treatment of inflammations.
Both enzymes were expressed in the E. coli strain C43(DE3) by
coexpression of the E. coli chaperones GroEL/GroES encoded in the
vector pGro12. These chaperones ensure a correct protein folding
which is important for an incorporation of the heme prosthetic
group. In FIG. 3 an SDS-PAGE and a CO difference spectrum of
purified bovine CYP21 are shown. As the CO difference spectrum
shows, the enzyme was purified in an active form. To determine the
binding of medrane to both isozymes the binding constants
(K.sub.D-values) were determined.
1.2 Expression of Electron Delivering Redox Partners
For an efficient substrate conversion, both isoforms require an
electron transfer system which consists of two parts, the
cytochrome P450 enzyme itself and one or two electron transfer
proteins which are essential for a hydroxylation reaction. Without
these transfer proteins, no reaction will take place. Electrons can
be transferred to CYP21 for example by the six electron transfer
systems listed in Table 2:
TABLE-US-00002 TABLE 2 Electron delivering proteins applied in
CYP21-dependent substrate conversions and corresponding expression
plasmids for whole-cell systems. hCYP21 or bCYP21 were combined in
reconstituted systems or whole-cell systems with the indicated
redox partners bCPR (bovine NADPH-dependent cytochrome P450
reductase), bAdR (bovine adrenodoxin reductase), bAdx.sub.4-108
(bovine adrenodoxin), Fpr (E. coli flavodoxin reductase), Arh1 (S.
pombe adrenodoxin reductase homolog), etp1.sup.fd (S. pombe
electron transfer protein, ferredoxin domain). Protein combinations
in reconstituted in vitro systems Corresponding plasmids Reductase
Ferredoxin in whole-cell systems 1 CPR p21h_bRED/p21b_bRED 2 AdR
Adx.sub.4-108 p21h_AdAx/p21b_AdAx 3 Fpr Adx.sub.4-108
p21h_FrAx/p21b_FrAx 4 Arh Adx.sub.4-108 p21h_ArAx/p21b_ArAx 5 Arh
etp1.sup.fd p21h_ArET/p21b_ArET 6 Fpr etp1.sup.fd
p21h_FrET/p21b_FrET
For in vitro studies and a verification of a substrate conversion,
all redox partners were purified.
1.3 Reconstitution of Cytochrome P450 Systems In Vitro
In vitro substrate conversions with purified enzymes in a defined
buffer and with an NADPH regeneration system revealed that both
isoforms together with the here listed electron transfer proteins
are able to convert medrane to premedrol very efficiently. FIG. 4
shows the in vitro conversion of medrane by human CYP21 together
with electron transfer system 2. This result indicates that
steroids as exemplified by premedrol can be produced enzymatically
by CYP21 together with a suitable redox system, e.g. as shown in
Table 2.
Example 2: Whole-Cell Systems
In view of the successful in vitro conversion of steroids, a
biotransformation in whole cells was developed.
Generally, in order to perform the hydroxylation in whole cells,
the CYP21 as well as the necessary electron transfer proteins were
expressed heterologously in Escherichia coli strain C43(DE3). For
expression and following conversion, bi- or tricistronic vectors
based on the plasmid pET17b were constructed, which carry the genes
for the particular CYP21 and the particular redox system. FIG. 5
shows all constructed vectors. To facilitate correct protein
folding, the E. coli chaperones GroEL and GroES were co-expressed
on a second vector. The transformed E. coli cells were able to
produce the CYP21 enzyme as well as the needed redox partners.
After the protein expression, a substrate conversion took place
which was started by the addition of the steroid to be hydroxylated
(exemplified by medrane) as a substrate.
In particular, E. coli strain C43(DE3) was transformed with vector
for whole cell biocatalysis (e.g. p21b_ArET) and the pGro12 which
encodes the chaperones GroEL/ES. The culture comprised 200 mL TB
medium (+antibiotics ampicillin and kanamycin) in a 2 L Erlenmeyer
flask, inoculated with 2 mL seed culture, and was grown at
37.degree. C. Expression was induced at OD 0.5 by addition of 1 mM
IPTG, 1 mM .delta.-aminolevulinic acid, 4 mg/mL arabinose and
maintained for 28 h at 27.degree. C. For whole cell
biotransformation, cells were harvested by centrifugation and
washed with 50 mM potassium phosphate buffer (pH 7.4). Substrate
conversion was started with the addition of 400 .mu.M substrate
with resting cells in 25 mL potassium phosphate buffer (50 mM)
including 2% glycerol, 1 mM IPTG, 1 mM .delta.-aminolevulinic acid,
4 mg/mL arabinose in 300 mL buffled flasks for 24 h at a cell
density of ca. 24 g/L (wet weight). Samples were taken after, e.g.,
24 h and measurement was performed via RP-HPLC after steroid
extraction with chloroform.
FIG. 6 shows that the steroid was converted to its 21-hydroxylated
derivative and that the appearance of by-products was not observed,
in contrast to a chemical synthesis. Time-dependent product
formation was studied in whole cells with the six redox systems for
each CYP21 isoform to determine not only an endpoint yield but also
the velocity of the reaction which is of high interest regarding a
biotechnological process (FIG. 7).
Next to the medrane-to-premedrol conversion, both human and bovine
CYP21 were able to hydroxylate all tested 3-ketosteroids which are
not yet hydroxylated at position 21. In particular, the following
steroid conversions could be shown: Medrane to premedrol
(non-natural substrate) Deltamedrane to medrol (non-natural
substrate) Progesterone to 11-deoxycorticosterone (natural
substrate) 170H-progesterone to 11-deoxycortisol (natural
substrate) Medroxyprogesterone to 21OH-medroxyprogesterone
(non-natural substrate) 5.alpha.-dihydroprogesterone to
21OH-(5.alpha.-dihydroprogesterone).
SEQUENCE LISTINGS
1
41495PRTHomo sapiens 1Met Leu Leu Leu Gly Leu Leu Leu Leu Leu Pro
Leu Leu Ala Gly Ala1 5 10 15Arg Leu Leu Trp Asn Trp Trp Lys Leu Arg
Ser Leu His Leu Pro Pro 20 25 30Leu Ala Pro Gly Phe Leu His Leu Leu
Gln Pro Asp Leu Pro Ile Tyr 35 40 45Leu Leu Gly Leu Thr Gln Lys Phe
Gly Pro Ile Tyr Arg Leu His Leu 50 55 60Gly Leu Gln Asp Val Val Val
Leu Asn Ser Lys Arg Thr Ile Glu Glu65 70 75 80Ala Met Val Lys Lys
Trp Ala Asp Phe Ala Gly Arg Pro Glu Pro Leu 85 90 95Thr Tyr Lys Leu
Val Ser Arg Asn Tyr Pro Asp Leu Ser Leu Gly Asp 100 105 110Tyr Ser
Leu Leu Trp Lys Ala His Lys Lys Leu Thr Arg Ser Ala Leu 115 120
125Leu Leu Gly Ile Arg Asp Ser Met Glu Pro Val Val Glu Gln Leu Thr
130 135 140Gln Glu Phe Cys Glu Arg Met Arg Ala Gln Pro Gly Thr Pro
Val Ala145 150 155 160Ile Glu Glu Glu Phe Ser Leu Leu Thr Cys Ser
Ile Ile Cys Tyr Leu 165 170 175Thr Phe Gly Asp Lys Ile Lys Asp Asp
Asn Leu Met Pro Ala Tyr Tyr 180 185 190Lys Cys Ile Gln Glu Val Leu
Lys Thr Trp Ser His Trp Ser Ile Gln 195 200 205Ile Val Asp Val Ile
Pro Phe Leu Arg Phe Phe Pro Asn Pro Gly Leu 210 215 220Arg Arg Leu
Lys Gln Ala Ile Glu Lys Arg Asp His Ile Val Glu Met225 230 235
240Gln Leu Arg Gln His Lys Glu Ser Leu Val Ala Gly Gln Trp Arg Asp
245 250 255Met Met Asp Tyr Met Leu Gln Gly Val Ala Gln Pro Ser Met
Glu Glu 260 265 270Gly Ser Gly Gln Leu Leu Glu Gly His Val His Met
Ala Ala Val Asp 275 280 285Leu Leu Ile Gly Gly Thr Glu Thr Thr Ala
Asn Thr Leu Ser Trp Ala 290 295 300Val Val Phe Leu Leu His His Pro
Glu Ile Gln Gln Arg Leu Gln Glu305 310 315 320Glu Leu Asp His Glu
Leu Gly Pro Gly Ala Ser Ser Ser Arg Val Pro 325 330 335Tyr Lys Asp
Arg Ala Arg Leu Pro Leu Leu Asn Ala Thr Ile Ala Glu 340 345 350Val
Leu Arg Leu Arg Pro Val Val Pro Leu Ala Leu Pro His Arg Thr 355 360
365Thr Arg Pro Ser Ser Ile Ser Gly Tyr Asp Ile Pro Glu Gly Thr Val
370 375 380Ile Ile Pro Asn Leu Gln Gly Ala His Leu Asp Glu Thr Val
Trp Glu385 390 395 400Arg Pro His Glu Phe Trp Pro Asp Arg Phe Leu
Glu Pro Gly Lys Asn 405 410 415Ser Arg Ala Leu Ala Phe Gly Cys Gly
Ala Arg Val Cys Leu Gly Glu 420 425 430Pro Leu Ala Arg Leu Glu Leu
Phe Val Val Leu Thr Arg Leu Leu Gln 435 440 445Ala Phe Thr Leu Leu
Pro Ser Gly Asp Ala Leu Pro Ser Leu Gln Pro 450 455 460Leu Pro His
Cys Ser Val Ile Leu Lys Met Gln Pro Phe Gln Val Arg465 470 475
480Leu Gln Pro Arg Gly Met Gly Ala His Ser Pro Gly Gln Ser Gln 485
490 4952496PRTBos taurus 2Met Val Leu Ala Gly Leu Leu Leu Leu Leu
Thr Leu Leu Ala Gly Ala1 5 10 15His Leu Leu Trp Gly Arg Trp Lys Leu
Arg Asn Leu His Leu Pro Pro 20 25 30Leu Val Pro Gly Phe Leu His Leu
Leu Gln Pro Asn Leu Pro Ile His 35 40 45Leu Leu Ser Leu Thr Gln Lys
Leu Gly Pro Val Tyr Arg Leu Arg Leu 50 55 60Gly Leu Gln Glu Val Val
Val Leu Asn Ser Lys Arg Thr Ile Glu Glu65 70 75 80Ala Met Ile Arg
Lys Trp Val Asp Phe Ala Gly Arg Pro Gln Ile Pro 85 90 95Ser Tyr Lys
Leu Val Ser Gln Arg Cys Gln Asp Ile Ser Leu Gly Asp 100 105 110Tyr
Ser Leu Leu Trp Lys Ala His Lys Lys Leu Thr Arg Ser Ala Leu 115 120
125Leu Leu Gly Thr Arg Ser Ser Met Glu Pro Trp Val Asp Gln Leu Thr
130 135 140Gln Glu Phe Cys Glu Arg Met Arg Val Gln Ala Gly Ala Pro
Val Thr145 150 155 160Ile Gln Lys Glu Phe Ser Leu Leu Thr Cys Ser
Ile Ile Cys Tyr Leu 165 170 175Thr Phe Gly Asn Lys Glu Asp Thr Leu
Val His Ala Phe His Asp Cys 180 185 190Val Gln Asp Leu Met Lys Thr
Trp Asp His Trp Ser Ile Gln Ile Leu 195 200 205Asp Met Val Pro Phe
Leu Arg Phe Phe Pro Asn Pro Gly Leu Trp Arg 210 215 220Leu Lys Gln
Ala Ile Glu Asn Arg Asp His Met Val Glu Lys Gln Leu225 230 235
240Thr Arg His Lys Glu Ser Met Val Ala Gly Gln Trp Arg Asp Met Thr
245 250 255Asp Tyr Met Leu Gln Gly Val Gly Arg Gln Arg Val Glu Glu
Gly Pro 260 265 270Gly Gln Leu Leu Glu Gly His Val His Met Ser Val
Val Asp Leu Phe 275 280 285Ile Gly Gly Thr Glu Thr Thr Ala Ser Thr
Leu Ser Trp Ala Val Ala 290 295 300Phe Leu Leu His His Pro Glu Ile
Gln Arg Arg Leu Gln Glu Glu Leu305 310 315 320Asp Arg Glu Leu Gly
Pro Gly Ala Ser Cys Ser Arg Val Thr Tyr Lys 325 330 335Asp Arg Ala
Arg Leu Pro Leu Leu Asn Ala Thr Ile Ala Glu Val Leu 340 345 350Arg
Leu Arg Pro Val Val Pro Leu Ala Leu Pro His Arg Thr Thr Arg 355 360
365Pro Ser Ser Ile Phe Gly Tyr Asp Ile Pro Glu Gly Met Val Val Ile
370 375 380Pro Asn Leu Gln Gly Ala His Leu Asp Glu Thr Val Trp Glu
Gln Pro385 390 395 400His Glu Phe Arg Pro Asp Arg Phe Leu Glu Pro
Gly Ala Asn Pro Ser 405 410 415Ala Leu Ala Phe Gly Cys Gly Ala Arg
Val Cys Leu Gly Glu Ser Leu 420 425 430Ala Arg Leu Glu Leu Phe Val
Val Leu Leu Arg Leu Leu Gln Ala Phe 435 440 445Thr Leu Leu Pro Pro
Pro Val Gly Ala Leu Pro Ser Leu Gln Pro Asp 450 455 460Pro Tyr Cys
Gly Val Asn Leu Lys Val Gln Pro Phe Gln Val Arg Leu465 470 475
480Gln Pro Arg Gly Val Glu Ala Gly Ala Trp Glu Ser Ala Ser Ala Gln
485 490 4953481PRTHomo sapiens 3Met Ala Lys Lys Thr Ser Ser Lys Gly
Lys Pro Pro Leu Ala Pro Gly1 5 10 15Phe Leu His Leu Leu Gln Pro Asp
Leu Pro Ile Tyr Leu Leu Gly Leu 20 25 30Thr Gln Lys Phe Gly Pro Ile
Tyr Arg Leu His Leu Gly Leu Gln Asp 35 40 45Val Val Val Leu Asn Ser
Lys Arg Thr Ile Glu Glu Ala Met Val Lys 50 55 60Lys Trp Ala Asp Phe
Ala Gly Arg Pro Glu Pro Leu Thr Tyr Lys Leu65 70 75 80Val Ser Arg
Asn Tyr Pro Asp Leu Ser Leu Gly Asp Tyr Ser Leu Leu 85 90 95Trp Lys
Ala His Lys Lys Leu Thr Arg Ser Ala Leu Leu Leu Gly Ile 100 105
110Arg Asp Ser Met Glu Pro Val Val Glu Gln Leu Thr Gln Glu Phe Cys
115 120 125Glu Arg Met Arg Ala Gln Pro Gly Thr Pro Val Ala Ile Glu
Glu Glu 130 135 140Phe Ser Leu Leu Thr Cys Ser Ile Ile Cys Tyr Leu
Thr Phe Gly Asp145 150 155 160Lys Ile Lys Asp Asp Asn Leu Met Pro
Ala Tyr Tyr Lys Cys Ile Gln 165 170 175Glu Val Leu Lys Thr Trp Ser
His Trp Ser Ile Gln Ile Val Asp Val 180 185 190Ile Pro Phe Leu Arg
Phe Phe Pro Asn Pro Gly Leu Arg Arg Leu Lys 195 200 205Gln Ala Ile
Glu Lys Arg Asp His Ile Val Glu Met Gln Leu Arg Gln 210 215 220His
Lys Glu Ser Leu Val Ala Gly Gln Trp Arg Asp Met Met Asp Tyr225 230
235 240Met Leu Gln Gly Val Ala Gln Pro Ser Met Glu Glu Gly Ser Gly
Gln 245 250 255Leu Leu Glu Gly His Val His Met Ala Ala Val Asp Leu
Leu Ile Gly 260 265 270Gly Thr Glu Thr Thr Ala Asn Thr Leu Ser Trp
Ala Val Val Phe Leu 275 280 285Leu His His Pro Glu Ile Gln Gln Arg
Leu Gln Glu Glu Leu Asp His 290 295 300Glu Leu Gly Pro Gly Ala Ser
Ser Ser Arg Val Pro Tyr Lys Asp Arg305 310 315 320Ala Arg Leu Pro
Leu Leu Asn Ala Thr Ile Ala Glu Val Leu Arg Leu 325 330 335Arg Pro
Val Val Pro Leu Ala Leu Pro His Arg Thr Thr Arg Pro Ser 340 345
350Ser Ile Ser Gly Tyr Asp Ile Pro Glu Gly Thr Val Ile Ile Pro Asn
355 360 365Leu Gln Gly Ala His Leu Asp Glu Thr Val Trp Glu Arg Pro
His Glu 370 375 380Phe Trp Pro Asp Arg Phe Leu Glu Pro Gly Lys Asn
Ser Arg Ala Leu385 390 395 400Ala Phe Gly Cys Gly Ala Arg Val Cys
Leu Gly Glu Pro Leu Ala Arg 405 410 415Leu Glu Leu Phe Val Val Leu
Thr Arg Leu Leu Gln Ala Phe Thr Leu 420 425 430Leu Pro Ser Gly Asp
Ala Leu Pro Ser Leu Gln Pro Leu Pro His Cys 435 440 445Ser Val Ile
Leu Lys Met Gln Pro Phe Gln Val Arg Leu Gln Pro Arg 450 455 460Gly
Met Gly Ala His Ser Pro Gly Gln Ser Gln His His His His His465 470
475 480His4482PRTbos taurus 4Met Ala Lys Lys Thr Ser Ser Lys Gly
Lys Pro Pro Leu Val Pro Gly1 5 10 15Phe Leu His Leu Leu Gln Pro Asn
Leu Pro Ile His Leu Leu Ser Leu 20 25 30Thr Gln Lys Leu Gly Pro Val
Tyr Arg Leu Arg Leu Gly Leu Gln Glu 35 40 45Val Val Val Leu Asn Ser
Lys Arg Thr Ile Glu Glu Ala Met Ile Arg 50 55 60Lys Trp Val Asp Phe
Ala Gly Arg Pro Gln Ile Pro Ser Tyr Lys Leu65 70 75 80Val Ser Gln
Arg Cys Gln Asp Ile Ser Leu Gly Asp Tyr Ser Leu Leu 85 90 95Trp Lys
Ala His Lys Lys Leu Thr Arg Ser Ala Leu Leu Leu Gly Thr 100 105
110Arg Ser Ser Met Glu Pro Trp Val Asp Gln Leu Thr Gln Glu Phe Cys
115 120 125Glu Arg Met Arg Val Gln Ala Gly Ala Pro Val Thr Ile Gln
Lys Glu 130 135 140Phe Ser Leu Leu Thr Cys Ser Ile Ile Cys Tyr Leu
Thr Phe Gly Asn145 150 155 160Lys Glu Asp Thr Leu Val His Ala Phe
His Asp Cys Val Gln Asp Leu 165 170 175Met Lys Thr Trp Asp His Trp
Ser Ile Gln Ile Leu Asp Met Val Pro 180 185 190Phe Leu Arg Phe Phe
Pro Asn Pro Gly Leu Trp Arg Leu Lys Gln Ala 195 200 205Ile Glu Asn
Arg Asp His Met Val Glu Lys Gln Leu Thr Arg His Lys 210 215 220Glu
Ser Met Val Ala Gly Gln Trp Arg Asp Met Thr Asp Tyr Met Leu225 230
235 240Gln Gly Val Gly Arg Gln Arg Val Glu Glu Gly Pro Gly Gln Leu
Leu 245 250 255Glu Gly His Val His Met Ser Val Val Asp Leu Phe Ile
Gly Gly Thr 260 265 270Glu Thr Thr Ala Ser Thr Leu Ser Trp Ala Val
Ala Phe Leu Leu His 275 280 285His Pro Glu Ile Gln Arg Arg Leu Gln
Glu Glu Leu Asp Arg Glu Leu 290 295 300Gly Pro Gly Ala Ser Cys Ser
Arg Val Thr Tyr Lys Asp Arg Ala Arg305 310 315 320Leu Pro Leu Leu
Asn Ala Thr Ile Ala Glu Val Leu Arg Leu Arg Pro 325 330 335Val Val
Pro Leu Ala Leu Pro His Arg Thr Thr Arg Pro Ser Ser Ile 340 345
350Phe Gly Tyr Asp Ile Pro Glu Gly Met Val Val Ile Pro Asn Leu Gln
355 360 365Gly Ala His Leu Asp Glu Thr Val Trp Glu Gln Pro His Glu
Phe Arg 370 375 380Pro Asp Arg Phe Leu Glu Pro Gly Ala Asn Pro Ser
Ala Leu Ala Phe385 390 395 400Gly Cys Gly Ala Arg Val Cys Leu Gly
Glu Ser Leu Ala Arg Leu Glu 405 410 415Leu Phe Val Val Leu Leu Arg
Leu Leu Gln Ala Phe Thr Leu Leu Pro 420 425 430Pro Pro Val Gly Ala
Leu Pro Ser Leu Gln Pro Asp Pro Tyr Cys Gly 435 440 445Val Asn Leu
Lys Val Gln Pro Phe Gln Val Arg Leu Gln Pro Arg Gly 450 455 460Val
Glu Ala Gly Ala Trp Glu Ser Ala Ser Ala Gln His His His His465 470
475 480His His
* * * * *
References