U.S. patent application number 10/623292 was filed with the patent office on 2005-04-14 for modified enzymes having polymer conjugates.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Olsen, Arne Agerlin, Roggen, Erwin Ludo, von der Osten, Claus.
Application Number | 20050079593 10/623292 |
Document ID | / |
Family ID | 8090149 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050079593 |
Kind Code |
A1 |
von der Osten, Claus ; et
al. |
April 14, 2005 |
Modified enzymes having polymer conjugates
Abstract
The present invention relates to polypeptide-polymer conjugates
having added and/or removed one or more attachment groups for
coupling polymeric molecules on the surface of the polypeptide
structure, a method for preparing polypeptide-polymer conjugates of
the invention, the use of said conjugated for reducing the
immunogenicity and allergenicity and compositions comprising said
conjugate.
Inventors: |
von der Osten, Claus;
(Lyngby, DK) ; Olsen, Arne Agerlin; (Virum,
DK) ; Roggen, Erwin Ludo; (Lyngby, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
DK-2880
|
Family ID: |
8090149 |
Appl. No.: |
10/623292 |
Filed: |
July 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10623292 |
Jul 18, 2003 |
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09705185 |
Nov 2, 2000 |
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6623950 |
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09705185 |
Nov 2, 2000 |
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09024532 |
Feb 17, 1998 |
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6245901 |
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09024532 |
Feb 17, 1998 |
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PCT/DK98/00046 |
Feb 6, 1998 |
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Current U.S.
Class: |
435/183 ;
530/350 |
Current CPC
Class: |
A61K 47/56 20170801;
C11D 3/386 20130101; A61K 47/60 20170801; C11D 3/37 20130101 |
Class at
Publication: |
435/183 ;
530/350 |
International
Class: |
C12N 009/00; C07K
014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 1997 |
DK |
0135/97 |
Claims
1-35. (canceled)
36. A conjugate comprising a lipase moiety conjugated to one or
more polymers, wherein the lipase moiety is a Humicola lanuginosa
lipase which comprises one or more of the following substitutions:
A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K,
G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K,
G106K, R118K, V120K, R125K, R133K, P136K, R139K, R160K, R179K,
R209K, G225K, L227K, V228K, P229K, P250K and F262K.
37. The conjugate of claim 36, wherein the Humicola lanuginosa
lipase has an amino acid sequence of SEQ ID NO: 6.
38. The conjugate of claim 36, wherein the polymer(s) have a
molecular weight from 1 to 60 kDa.
39. The conjugate of claim 36, wherein the polymer(s) are natural
or synthetic homo or heteropolymers.
40. The conjugate of claim 36, wherein the polymer(s) are selected
from the group consisting of polyols, polyamines, polycarboxyl
acids and polymers comprising a hydroxyl group and an amine
group.
41. The conjugate of claim 36, wherein the polymer(s) are selected
from the group consisting of polyalkylene oxides (PAO),
PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),
branched PEGs, polyvinyl alcohols (PVA), poly-carboxylates,
polyvinylpyrolidones, poly-D,L-amino acids, polyethylene-co-maleic
acid anhydride, polystyrene-co-malic acid anhydrides, dextrans,
heparins, homologous albumins, celluloses, hydrolysates of
chitosan, starches, glycogen, agaroses and derivatives thereof,
guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin,
alginic acid hydrolysates and bio-polymers.
42. The conjugate of claim 36, wherein the polymer(s) are
polyalkylene glycols (PAG) or methoxypolyethylene glycols
(mPEG).
43. The conjugate of claim 36, wherein the polymer(s) are selected
from the group consisting of polyethylene glycols (PEG),
polypropylene glycols and carboxymethyl-dextrans.
44. The conjugate of claim 36, wherein the polymer(s) are selected
from the group consisting of methylcellulose,
carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose
carboxyethylcellulose and hydroxypropylcellulose.
45. The conjugate of claim 36, wherein the polymer(s) are
hydroxyethyl-starches or hydroxypropyl-starches.
46. The conjugate of claim 36, wherein the polymer(s) are
methoxypolyethylene glycols (mPEG).
47. A detergent composition comprising a conjugate of claim 36 and
a surfactant.
48. A skin care composition, comprising a conjugate of claim 36 and
ingredients used in skin care products.
49. A pharmaceutical composition comprising a conjugate of claim 36
and further comprising ingredients used in pharmaceuticals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is divisional of application Ser. No.
09/705,185 filed Nov. 2, 2000, which is a divisional of application
Ser. No. 09/024,532 filed Feb. 17, 1998 which is a continuation of
PCT/DK98/00046 filed Feb. 6, 1998, which claims priority under 35
U.S.C. 119 of Danish application no. 0135/97 filed Feb. 6,
1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to polypeptide-polymer
conjugates having added and/or removed one or more attachment
groups for coupling polymeric molecules on the surface of the 3D
structure of the polypeptide, a method for preparing
polypeptide-polymer conjugates of the invention, the use of said
conjugated for reducing the immunogenicity and allergenicity, and
compositions comprising said conjugate.
[0004] 2. Description of Related Art
[0005] The use of polypeptides, including enzymes, in the
circulatory system to obtain a particular physiological effect is
well-known in the medical arts. Further, within the arts of
industrial applications, such as laundry washing, textile
bleaching, person care, contact lens cleaning, food and feed
preparation enzymes are used as a functional ingredient. One of the
important differences between pharmaceutical and industrial
application is that for the latter type of applications (i.e.
industrial applications) the polypeptides (often enzymes) are not
intended to enter into the circulatory system of the body.
[0006] Certain polypeptides and enzymes have an unsatisfactory
stability and may under certain circumstances--dependent on the way
of challenge--cause an immune response, typically an IgG and/or IgE
response.
[0007] It is today generally recognized that the stability of
polypeptides is improved and the immune response is reduced when
polypeptides, such as enzymes, are coupled to polymeric molecules.
It is believed that the reduced immune response is a result of the
shielding of (the) epitope(s) on the surface of the polypeptide
responsible for the immune response leading to antibody formation
by the coupled polymeric molecules.
[0008] Techniques for conjugating polymeric molecules to
polypeptides are well-known in the art.
[0009] One of the first commercially suitable techniques was
described back in the early 1970's and disclosed in e.g. U.S. Pat.
No. 4,179,337. Said patent concerns non-immunogenic polypeptides,
such as enzymes and peptide hormones coupled to polyethylene glycol
(PEG) or polypropylene glycol (PPG). At least 15% of polypeptides'
physiological activity is maintained.
[0010] GB patent no. 1,183,257 (Crook et al.) describes chemistry
for conjugation of enzymes to polysaccharides via a triazine
ring.
[0011] Further, techniques for maintaining of the enzymatic
activity of enzyme-polymer conjugates are also known in the
art.
[0012] WO 93/15189 (Veronese et al.) concerns a method for
maintaining the activity in polyethylene glycol-modified
proteolytic enzymes by linking the proteolytic enzyme to a
macromolecularized inhibitor. The conjugates are intended for
medical applications.
[0013] It has been found that the attachment of polymeric molecules
to a polypeptide often has the effect of reducing the activity of
the polypeptide by interfering with the interaction between the
polypeptide and its substrate. EP 183 503 (Beecham Group PLC)
discloses a development of the above concept by providing
conjugates comprising pharmaceutically useful proteins linked to at
least one water-soluble polymer by means of a reversible linking
group.
[0014] EP 471,125 (Kanebo) discloses skin care products comprising
a parent protease (Bacillus protease with the trade name
Esperase.RTM.) coupled to polysaccharides through a triazine ring
to improve the thermal and preservation stability. The coupling
technique used is also described in the above mentioned GB patent
no. 1,183,257 (Crook et al.).
[0015] JP 3083908 describes a skin cosmetic material which contains
a transglutaminase from guinea pig liver modified with one or more
water-soluble substances such as PEG, starch, cellulose etc. The
modification is performed by activating the polymeric molecules and
coupling them to the enzyme. The composition is stated to be mild
to the skin.
[0016] However, it is not always possible to readily couple
polymeric molecules to polypeptides and enzymes. Further, there is
still a need for polypeptide-polymer conjugates with an even more
reduced immunogenicity and/or allergenicity.
SUMMARY OF THE INVENTION
[0017] It is the object of the present invention to provide
improved polypeptide-polymer conjugates suitable for industrial and
pharmaceutical applications.
[0018] The term "improved polypeptide-polymer conjugates" means in
the context of the present invention conjugates having a reduced
immune response in humans and animals and/or an improved stability.
As will be described further below the immune response is dependent
on the way of challenge.
[0019] The present inventors have found that polypeptides, such as
enzymes, may be made less immunogenic and/or allergenic by adding
and/or removing one or more attachment groups on the surface of the
parent polypeptide to be coupled to polymeric molecules.
[0020] When introducing pharmaceutical polypeptide directly into
the circulatory system (i.e. bloodstream) the potential risk is an
immunogenic response in the form of mainly IgG, IgA and/or IgM
antibodies. In contrast hereto, industrial polypeptides, such as
enzymes used as a functional ingredient in e.g. detergents, are not
intended to enter the circulatory system. The potential risk in
connection with industrial polypeptides is inhalation causing an
allergenic response in the form of mainly IgE antibody
formation.
[0021] Therefore, in connection with industrial polypeptides the
potential risk is respiratory allergenicity caused by inhalation,
intratracheal and intranasal presentation of polypeptides.
[0022] The main potential risk of pharmaceutical polypeptides is
immunogenicity caused by intradermal, intravenous or subcutaneous
presentation of the polypeptide.
[0023] It is to be understood that reducing the "immunogenicity"
and reducing the "respiratory allergenicity" are two very different
problems based on different routes of exposure and on two very
different immunological mechanisms:
[0024] The term "immunogenicity" used in connection with the
present invention may be referred to as allergic contact dermatitis
in a clinical setting and is a cell mediated delayed immune
response to chemicals that contact and penetrate the skin. This
cell mediated reaction is also termed delayed contact
hypersensitivity (type IV reaction according to Gell and Combs
classification of immune mechanisms in tissue damage).
[0025] The term "allergenicity" or "respiratory allergenicity" is
an immediate anaphylactic reaction (type I antibody-mediated
reaction according to Gell and Combs) following inhalation of e.g.
polypeptides.
[0026] According to the present invention it is possible to provide
polypeptides with a reduced immune response and/or improved
stability, which has a substantially retained residual
activity.
[0027] The allergic and the immunogenic response are in one term,
at least in the context of the present invention called the "immune
response".
[0028] In the first aspect the invention relates to a
polypeptide-polymer conjugate having
[0029] a) one or more additional polymeric molecules coupled to the
polypeptide having been modified in a manner to increase the number
of attachment groups on the surface of the polypeptide in
comparison to the number of attachment groups available on the
corresponding parent polypeptide, and/or
[0030] b) one or more fewer polymeric molecules coupled to the
polypeptide having been modified in a manner to decrease the number
of attachment groups at or close to the functional site(s) of the
polypeptide in comparison to the number of attachment groups
available on the corresponding parent polypeptide.
[0031] The term "parent polypeptide" refers to the polypeptide to
be modified by coupling to polymeric molecules. The parent
polypeptide may be a naturally-occurring (or wild-type) polypeptide
or may be a variant thereof prepared by any suitable means. For
instance, the parent polypeptide may be a variant of a
naturally-occurring polypeptide which has been modified by
substitution, deletion or truncation of one or more amino acid
residues or by addition or insertion of one or more amino acid
residues to the amino acid sequence of a naturally-occurring
polypeptide.
[0032] A "suitable attachment group" means in the context of the
present invention any amino acid residue group on the surface of
the polypeptide capable of coupling to the polymeric molecule in
question.
[0033] Preferred attachment groups are amino groups of lysine
residues and the N-terminal amino group. Polymeric molecules may
also be coupled to the carboxylic acid groups (--COOH) of amino
acid residues in the polypeptide chain located on the surface.
Carboxylic acid attachment groups may be the carboxylic acid group
of aspartate or glutamate and the C-terminal COOH-group.
[0034] A "functional site" means any amino acid residues and/or
cofactors which are known to be essential for the performance of
the polypeptide, such as catalytic activity, e.g. the catalytic
triad residues, histidine, aspartate and serine in serine
proteases, or e.g. the heme group and the distal and proximal
histidines in a peroxidase such as the Arthromyces ramosus
peroxidase.
[0035] In the second aspect the invention relates to a method for
preparing improved polypeptide-polymer conjugates comprising the
steps of:
[0036] a) identifying amino acid residues located on the surface of
the 3D structure of the parent polypeptide in question,
[0037] b) selecting target amino acid residues on the surface of
said 3D structure of said parent polypeptide to be mutated,
[0038] c) i) substituting or inserting one or more amino acid
residues selected in step b) with an amino acid residue having a
suitable attachment group, and/or ii) substituting or deleting one
or more amino acid residues selected in step b) at or close to the
functional site(s),
[0039] d) coupling polymeric molecules to the mutated
polypeptide.
[0040] The invention also relates to the use of a conjugate of the
invention and the method of the invention for reducing the
immunogenicity of pharmaceuticals and reducing the allergenicity of
industrial products.
[0041] Finally the invention relates to compositions comprising a
conjugate of the invention and further ingredients used in
industrial products or pharmaceuticals.
BRIEF DESCRIPTION OF THE DRAWING
[0042] FIG. 1 shows the anti-lipase serum antibody levels after 5
weekly immunizations with i) control ii) unmodified lipase variant,
iii) lipase variant-SPEG. (X: log(serum dilution); Y Optical
Density (490/620)).
DETAILED DESCRIPTION OF THE INVENTION
[0043] It is the object of the present invention to provide
improved polypeptide-polymer conjugates suitable for industrial and
pharmaceutical applications.
[0044] Even though polypeptides used for pharmaceutical
applications and industrial application can be quite different the
principle of the present invention may be tailored to the specific
type of parent polypeptide (i.e. enzyme, hormone peptides
etc.).
[0045] The inventors of the present invention have provided
improved polypeptide-polymer conjugates with a reduced immune
response in comparison to conjugates prepared from the
corresponding parent polypeptides.
[0046] The present inventors have found that polypeptides, such as
enzymes, may be made less immunogenic and/or less allergenic by
adding one or more attachment groups on the surface of the parent
polypeptide. In addition thereto the inventors have found that a
higher percentage of maintained residual functional activity may be
obtained by removing attachment groups at or close to the
functional site(s).
[0047] In the first aspect the invention relates to an improved
polypeptide-polymer conjugate having
[0048] a) one or more additional polymeric molecules coupled to the
polypeptide having been modified in a manner to increase the number
of attachment groups on the surface of the polypeptide in
comparison to the number of attachment groups available on the
corresponding parent polypeptide, and/or
[0049] b) one or more fewer polymeric molecules coupled to the
polypeptide having been modified in a manner to decrease the number
of attachment groups at or close to the functional site(s) of the
polypeptide in comparison to the number of attachment groups
available on the corresponding parent polypeptide.
[0050] Whether the attachment groups should be added and/or removed
depends on the specific parent polypeptide.
[0051] a) Addition of Attachment Groups
[0052] There may be a need for further attachment groups on the
polypeptide if only few attachment groups are available on the
surface of the parent polypeptide. The addition of one or more
attachment groups by substituting or inserting one or more amino
acid residues on the surface of the parent polypeptide increases
the number of polymeric molecules which may be attached in
comparison to the corresponding parent polypeptide. Conjugates with
an increased number of polymeric molecules attached thereto are
generally seen to have a reduced immune response in comparison to
the corresponding conjugates having fewer polymeric molecules
coupled thereto.
[0053] Any available amino acid residues on the surface of the
polypeptide, preferentially not being at or close to the functional
site(s), such as the active site(s) of enzymes, may in principle be
subject to substitution and/or insertion to provide additional
attachment groups.
[0054] As will be described further below the location of the
additional coupled polymeric molecules may be of importance for the
reduction of the immune response and the percentage of maintained
residual functional activity of the polypeptide itself.
[0055] A conjugate of the invention may typically have from 1 to
25, preferentially 1 to 10 or more additional polymeric molecules
coupled to the surface of the polypeptide in comparison to the
number of polymeric molecules of a conjugate prepared on the basis
of the corresponding parent polypeptide.
[0056] However, the optimal number of attachment groups to be added
depends (at least partly) on the surface area (i.e. molecular
weight) of the parent polypeptide to be shielded by the coupled
polymeric molecules, and also on the number of already available
attachment groups on the parent polypeptide.
[0057] b) Removing Attachment Groups
[0058] In the case of enzymes or other polypeptides performing
their function by interaction with a substrate or the like,
polymeric molecules coupled to the polypeptide might be impeded by
the interaction between the polypeptide and its substrate or the
like, if they are coupled at or close to the functional site(s)
(i.e. active site of enzymes). This will most probably cause
reduced activity.
[0059] In the case of enzymes having one or more polymeric
molecules coupled at or close to the active site a substantial loss
of residual enzymatic activity can be expected. Therefore,
according to the invention conjugates may be constructed to
maintain a higher percentage of residual enzymatic activity in
comparison to a corresponding conjugates prepared on the basis of
the parent enzyme in question. This may be done by substituting
and/or deleting attachment groups at or close to the active site,
hereby increasing the substrate affinity by improving the
accessibility of the substrate in the catalytic cleft.
[0060] An enzyme-polymer conjugate of the invention may typically
have from 1 to 25, preferably 1 to 10 fewer polymeric molecules
coupled at or close to the active site in comparison to the number
of polymeric molecules of a conjugate prepared on the basis of the
corresponding parent polypeptide.
[0061] As will be explained below "at or close to" the functional
site(s) means that no polymeric molecule(s) should be coupled
within 5 Angstroms, preferably 8 Angstroms, especially 10 Angstroms
of the functional site(s).
[0062] Removal of attachment groups at or close to the functional
site(s) of the polypeptide may advantageously be combined with
addition of attachment groups in other parts of the surface of the
polypeptide.
[0063] The total number of attachment groups may this way be
unchanged, increased or decreased. However the location(s) of the
total number of attachment group(s) is(are) improved assessed by
the reduction of the immune response and/or percentage of
maintained residual activity. Improved stability may also be
obtained this way.
[0064] The Number of Attachment Groups
[0065] Generally seen the number of attachment groups should be
balanced to the molecular weight and/or surface area of the
polypeptide. The more heavy the polypeptide is the more polymeric
molecules should be coupled to the polypeptide to obtain sufficient
shielding of the epitope(s) responsible for antibody formation.
[0066] Therefore, if the parent polypeptide molecule is relatively
light (e.g. 1 to 35 kDa) it may be advantageous to increase the
total number of coupled polymeric molecules (outside the functional
site(s)) to a total between 4 and 20.
[0067] If the parent polypeptide molecules are heavier, for
instance 35 to 60 kDa, the number of coupled polymeric molecules
(outside the functional site(s)) may advantageously be increased to
7 to 40, and so on.
[0068] The ratio between the molecular weight (Mw) of the
polypeptide in question and the number of coupled polymeric
molecules considered to be suitable by the inventors is listed
below in Table 1.
1 TABLE 1 Molecular weight of parent Number of polymeric molecules
polypeptide (M.sub.w) kDa coupled to the polypeptide 1 to 35 4-20
35 to 60 7-40 60 to 80 10-50 80 to 100 15-70 More than 100 more
than 20
[0069] Reduced Immune Response vs. Maintained Residual Enzymatic
Activity
[0070] Especially for enzymes, in comparison to many other types of
polypeptides, there is a conflict between reducing the immune
response and maintaining a substantial residual enzymatic activity
as the activity of enzymes are connected with interaction between a
substrate and the active site often present as a cleft in the
enzyme structure.
[0071] Without being limited to any theory it is believed that the
loss of enzymatic activity of enzyme-polymer conjugates might be a
consequence of impeded access of the substrate to the active site
in the form of spatial hindrance of the substrate by especially
bulky and/or heavy polymeric molecules to the catalytic cleft. It
might also, at least partly, be caused by disadvantageous minor
structural changes of the 3D structure of the enzyme due to the
stress made by the coupling of the polymeric molecules.
[0072] Maintained Residual Activity
[0073] A polypeptide-polymer conjugates of the invention has a
substantially maintained functional activity.
[0074] A "substantially" maintained functional activity is in the
context of the present invention defined as an activity which is at
least between 20% and 30%, preferably between 30% and 40%, more
preferably between 40% and 60%, better from 60% up to 80%, even
better from 80% up to about 100%, in comparison to the activity of
the conjugates prepared on the basis of corresponding parent
polypeptides.
[0075] In the case of polypeptide-polymer conjugates of the
invention where no polymeric molecules are coupled at or close to
the functional site(s) the residual activity may even be up to 100%
or very close thereto. If attachment group(s) of the parent
polypeptide is(are) removed from the functional site the activity
might even be more than 100% in comparison to modified (i.e.
polymer coupled) parent polypeptide conjugate.
[0076] Position of Coupled Polymeric Molecules
[0077] To obtain an optimally reduced immune response (i.e.
immunogenic and allergenic response) the polymeric molecules
coupled to the surface of the polypeptide in question should be
located in a suitable distance from each other.
[0078] In a preferred embodiment of the invention the parent
polypeptide is modified in a manner whereby the polymeric molecules
are spread broadly over the surface of the polypeptide. In the case
of the polypeptide in question has enzymatic activity it is
preferred to have as few as possible, especially none, polymeric
molecules coupled at or close to the area of the active site.
[0079] In the present context "spread broadly over the surface of
the polypeptide" means that the available attachment groups are
located so that the polymeric molecules shield different parts of
the surface, preferably the whole or close to the whole surface
area away from the functional site(s), to make sure that epitope(s)
are shielded and hereby not recognized by the immune system or its
antibodies.
[0080] The area of antibody-polypeptide interaction typically
covers an area of 500 Angstroms.sup.2, as described by Sheriff et
al., 1987, Proc. Natl. Acad. Sci. USA, 84, 8075-8079. 500
Angstroms.sup.2 corresponds to a rectangular box of 25
Angstroms.times.20 Angstroms or a circular region of radius 12.6
Angstroms. Therefore, to prevent binding of antibodies to the
epitope(s) to the polypeptide in question it is preferred to have a
maximum distance between two attachment groups around 10
Angstroms.
[0081] Consequently, amino acid residues which are located in
excess of 10 Angstroms away from already available attachment
groups are suitable target residues. If two or more attachment
groups on the polypeptide are located very close to each other it
will in most cases result in that only one polymeric molecule will
be coupled. To ensure a minimal loss of functional activity it is
preferred not to couple polymeric molecules at or close to the
functional site(s). Said distance depends at least partly on the
bulkiness of the polymeric molecules to be coupled, as impeded
access by the bulky polymeric molecules to the functional site is
undesired. Therefore, the more bulky the polymeric molecules are
the longer should the distance from the functional site to the
coupled polymeric molecules be.
[0082] To maintain a substantial functional activity of the
polypeptide in question attachment groups located within 5
Angstroms, preferred 8 Angstroms, especially 10 Angstroms from such
functional site(s) should be left uncoupled and may therefore
advantageously be removed or changed by mutation. Functional
residues should normally not be mutated/removed, even though they
potentially can be the target for coupling polymeric molecules. In
said case it may thus be advantageous to choose a coupling
chemistry involving different attachment groups.
[0083] Further, to provide a polypeptide having coupled polymeric
molecules at (a) known epitope(s) recognizable by the immune system
or close to said epitope(s) specific mutations at such sites are
also considered advantageous according to the invention. If the
position of the epitope(s) is(are) unknown it is advantageous to
couple several or many polymeric molecules to the polypeptide.
[0084] As also mentioned above it is preferred that said attachment
groups are spread broadly over the surface.
[0085] The Attachment Group
[0086] Virtually all ionized groups, such as the amino groups of
lysine residues, are located on the surface of the polypeptide
molecule (see for instance Thomas E. Creighton, 1993, "Proteins",
W.H. Freeman and Company, New York).
[0087] Therefore, the number of readily accessible attachment
groups (e.g. amino groups) on a modified or parent polypeptide
equals generally seen the number of lysine residues in the primary
structure of the polypeptide plus the N-terminus amino group.
[0088] The chemistry of coupling polymeric molecules to amino
groups are quite simple and well established in the art. Therefore,
it is preferred to add and/or remove lysine residues (i.e.
attachment groups) to/from the parent polypeptide in question to
obtain improved conjugates with reduced immunogenicity and/or
allergenicity and/or improved stability and/or high percentage
maintained functional activity.
[0089] Polymeric molecules may also be coupled to the carboxylic
groups (--COOH) of amino acid residues on the surface of the
polypeptide. Therefore, if using carboxylic groups (including the
C-terminal group) as attachment groups addition and/or removal of
aspartate and glutamate residues may also be suitable according to
the invention.
[0090] If using other attachment groups, such as --SH groups, they
may be added and/or removed analogously.
[0091] Substitution of the amino acid residues is preferred over
insertion, as the impact on the 3D structure of the polypeptide
normally will be less pronounced.
[0092] Preferred substitutions are conservative substitutions. In
the case of increasing the number of attachment groups the
substitution may advantageously be performed at a location having a
distance of 5 Angstroms, preferred 8 Angstroms, especially 10
Angstroms from the functional site(s) (active site for
enzymes).
[0093] An example of a suitable conservative substitution to obtain
an additional amino attachment group is an arginine to lysine
substitution. Examples of conservative substitutions to obtain
additional carboxylic attachment groups are aspargine to
aspartate/glutamate or glutamine to aspartate/glutamate
substitutions. To remove attachment groups a lysine residue may be
substituted with an arginine and so on.
[0094] The Parent Polypeptide
[0095] In the context of the present invention the term
"polypeptides" includes proteins, peptides and/or enzymes for
pharmaceutical or industrial applications. Typically the
polypeptides in question have a molecular weight in the range
between about 1 to 100 kDa, often 15 kDa and 100 kDa.
[0096] Pharmaceutical Polypeptides
[0097] The term "pharmaceutical polypeptides" is defined as
polypeptides, including peptides, such as peptide hormones,
proteins and/or enzymes, being physiologically active when
introduced into the circulatory system of the body of humans and/or
animals.
[0098] Pharmaceutical polypeptides are potentially immunogenic as
they are introduced into the circulatory system.
[0099] Examples of "pharmaceutical polypeptides" contemplated
according to the invention include insulin, ACTH, glucagon,
somatostatin, somatotropin, thymosin, parathyroid hormone,
pigmentary hormones, somatomedin, erythropoietin, luteinizing
hormone, chorionic gonadotropin, hypothalmic releasing factors,
antidiuretic hormones, thyroid stimulating hormone, relaxin,
interferon, thrombopoietin (TPO) and prolactin.
[0100] Industrial Polypeptides
[0101] Polypeptides used for industrial applications often have an
enzymatic activity. Industrial polypeptides (e.g. enzymes) are (in
contrast to pharmaceutical polypeptides) not intended to be
introduced into the circulatory system of the body.
[0102] It is not very like that industrial polypeptides, such as
enzymes used as ingredients in industrial compositions and/or
products, such as detergents and personal care products, including
cosmetics, come into direct contact with the circulatory system of
the body of humans or animals, as such enzymes (or products
comprising such enzymes) are not injected (or the like) into the
bloodstream.
[0103] Therefore, in the case of the industrial polypeptide the
potential risk is respiratory allergy (i.e. IgE response) as a
consequence of inhalation to polypeptides through the respiratory
passage.
[0104] In the context of the present invention "industrial
polypeptides" are defined as polypeptides, including peptides,
proteins and/or enzymes, which are not intended to be introduced
into the circulatory system of the body of humans and/or
animals.
[0105] Examples of such polypeptides are polypeptides, especially
enzymes, used in products such as detergents, household article
products, agrochemicals, personal care products, such as skin care
products, including cosmetics and toiletries, oral and dermal
pharmaceuticals, composition use for processing textiles,
compositions for hard surface cleaning, and compositions used for
manufacturing food and feed etc.
[0106] Enzymatic Activity
[0107] Pharmaceutical or industrial polypeptides exhibiting
enzymatic activity will often belong to one of the following groups
of enzymes including Oxidoreductases (E.C. 1, "Enzyme Nomenclature,
(1992), Academic Press, Inc.), such as laccase and Superoxide
dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases
(TGases); Hydrolases (E.C. 3), including proteases, especially
subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as
Protein disulfide Isomerases (PDI).
[0108] Hydrolases
[0109] Proteolytic Enzymes
[0110] Contemplated proteolytic enzymes include proteases selected
from the group of Aspartic proteases, such pepsins, cysteine
proteases, such as papain, serine proteases, such as subtilisins,
or metallo proteases, such as NEUTRASE.RTM..
[0111] Specific examples of parent proteases include PD498 (WO
93/24623 and SEQ ID NO: 2), SAVINASE.RTM. (von der Osten et al.,
1993, Journal of Biotechnology, 28, 55+, SEQ ID NO: 3), Proteinase
K (Gunkel et al., 1989, Eur. J. Biochem, 179, 185-194), Proteinase
R (Samal et al, 1990, Mol. Microbiol, 4, 1789-1792), Proteinase T
(Samal et al., 1989, Gene, 85, p. 329-333), Subtilisin DY (Betzel
et al. 1993, Arch. Biophys, 302(2), 499-502), Lion Y (JP
04197182-A), RENNILASE.RTM. (Available from Novo Nordisk A/S), JA16
(WO 92/17576), ALCALASE.RTM. (a natural subtilisin Carlberg
variant) (von der Osten et al., 1993, Journal of Biotechnology, 28,
55+).
[0112] Lipolytic Enzymes
[0113] Contemplated lipolytic enzymes include Humicola lanuginosa
lipases, e.g. the one described in EP 258 068 and EP 305 216 (See
SEQ ID NO: 6 below), Humicola insolens, a Rhizomucor miehei lipase,
e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO
96/13578), a Candida lipase, such as a C. antarctica lipase, e.g.
the C. antarctica lipase A or B described in EP 214 761, a
Pseudomonas lipase such as a P. alcaligenes and P.
pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P.
cepacia lipase, e.g. as described in EP 331 376, a Pseudomonas sp.
lipase as disclosed in WO 95/14783, a Bacillus lipase, e.g. a B.
subtilis lipase (Dartois et al., 1993 Biochemica et Biophysica Acta
1131, 253-260), a B. stearothermophilus lipase (JP 64/744992) and a
B. pumilus lipase (WO 91/16422). Other types of lipolytic enzymes
include cutinases, e.g. derived from Pseudomonas mendocina as
described in WO 88/09367, or a cutinase derived from Fusarium
solani pisi (e.g. described in WO 90/09446).
[0114] Oxidoreductases
[0115] Laccases
[0116] Contemplated laccases include Polyporus pinisitus laccase
(WO 96/00290), Myceliophthora laccase (WO 95/33836), Scytalidium
laccase (WO 95/338337), and Pyricularia oryzae laccase (Available
from Sigma).
[0117] Peroxidase
[0118] Contemplated peroxidases include B. pumilus peroxidases (WO
91/05858), Myxococcaceae peroxidase (WO 95/11964), Coprinus
cinereus (WO 95/10602) and Arthromyces ramosus peroxidase
(Kunishima et al. 1994, J. Mol. Biol., 235, 331-344).
[0119] Transferases
[0120] Transglutaminases
[0121] Suitable transferases include any transglutaminases
disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo
Nordisk A/S).
[0122] Isomerases
[0123] Protein Disulfide Isomerase
[0124] Without being limited thereto suitable protein disulfide
isomerases include PDIs described in WO 95/01425 (Novo Nordisk
A/S).
[0125] The Polymeric Molecule
[0126] The polymeric molecules coupled to the polypeptide may be
any suitable polymeric molecule, including natural and synthetic
homo-polymers, such as polyols (i.e. poly-OH), polyamines (i.e.
poly-NH.sub.2) and polycarboxyl acids (i.e. poly-COOH), and further
hetero-polymers i.e. polymers comprising one or more different
coupling groups e.g. a hydroxyl group and amine groups.
[0127] Examples of suitable polymeric molecules include polymeric
molecules selected from the group comprising polyalkylene oxides
(PAO), such as polyalkylene glycols (PAG), including polyethylene
glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene
glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole
(CDI-PEG), branched PEGs, polyvinyl alcohol (PVA),
polycarboxylates, polyvinylpyrolidones, poly-D,L-amino acids,
polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid
anhydride, dextrans including carboxymethyl-dextrans, heparin,
homologous albumin, celluloses, including methylcellulose,
carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose
carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of
chitosan, starches such as hydroxyethyl-starches and hydroxy
propyl-starches, glycogen, agaroses and derivatives thereof, guar
gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic
acid hydrolysates and bio-polymers.
[0128] Preferred polymeric molecules are non-toxic polymeric
molecules such as (m)polyethylene glycol ((m)PEG) which further
requires a relatively simple chemistry for its covalently coupling
to attachment groups on the enzyme's surface.
[0129] Generally seen polyalkylene oxides (PAO), such as
polyethylene oxides, such as PEG and especially mPEG, are the
preferred polymeric molecules, as these polymeric molecules, in
comparison to polysaccharides such as dextran, pullulan and the
like, have few reactive groups capable of cross-linking.
[0130] Even though all of the above mentioned polymeric molecules
may be used according to the invention the methoxypolyethylene
glycols (mPEG) may advantageously be used. This arises from the
fact that methoxyethylene glycols have only one reactive end
capable of conjugating with the enzyme. Consequently, the risk of
cross-linking is less pronounced. Further, it makes the product
more homogeneous and the reaction of the polymeric molecules with
the enzyme easier to control.
[0131] Preparation of Enzyme Variants
[0132] Enzyme variants to be conjugated may be constructed by any
suitable method. A number of methods are well established in the
art. For instance enzyme variants according to the invention may be
generated using the same materials and methods described in e.g. WO
89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870
(Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO
87/05050 (Genex), EP application no. 87303761 (Genentech), EP
260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578
(Genentech), WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164
(Genex), Thomas et al., 1985, Nature, 318, 375-376; Thomas et al.,
1987, J. Mol. Biol., 193, 803-813; Russel and Fersht, 1987, Nature,
328, 496-500.
[0133] Generation of Site Directed Mutations
[0134] Prior to mutagenesis the gene encoding the polypeptide of
interest must be cloned in a suitable vector. Methods for
generating mutations in specific sites are described below.
[0135] Once the polypeptide encoding gene has been cloned, and
desirable sites for mutation identified and the residue to
substitute for the original ones have been decided, these mutations
can be introduced using synthetic oligonucleotides. These
oligonucleotides contain nucleotide sequences flanking the desired
mutation sites; mutant nucleotides are inserted during
oligo-nucleotide synthesis. In a preferred method, Site-directed
mutagenesis is carried out by SOE-PCR mutagenesis technique
described by Kammann et al., 1989, Nucleic Acids Research, 17(13),
5404, and by Sarkar G. and Sommer, S. S., 1990, Biotechniques, 8,
404-407.
[0136] Activation of Polymers
[0137] If the polymeric molecules to be conjugated with the
polypeptide in question are not active it must be activated by the
use of a suitable technique. It is also contemplated according to
the invention to couple the polymeric molecules to the polypeptide
through a linker. Suitable linkers are well-known to the skilled
person.
[0138] Methods and chemistry for activation of polymeric molecules
as well as for conjugation of polypeptides are intensively
described in the literature. Commonly used methods for activation
of insoluble polymers include activation of functional groups with
cyanogen bromide, periodate, glutaraldehyde, biepoxides,
epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,
trichlorotriazine etc. (see R. F. Taylor, (1991), "Protein
Immobilisation. Fundamentals and Applications", Marcel Dekker,
N.Y.; S. S. Wong, 1992, "Chemistry of Protein Conjugation and
Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al., 1993,
"Immobilized Affinity Ligand Techniques", Academic Press, N.Y.).
Some of the methods concern activation of insoluble polymers but
are also applicable to activation of soluble polymers e.g.
periodate, trichlorotriazine, sulfonylhalides, divinylsulfone,
carbodiimide etc. The functional groups being amino, hydroxyl,
thiol, carboxyl, aldehyde or sulfydryl on the polymer and the
chosen attachment group on the protein must be considered in
choosing the activation and conjugation chemistry which normally
consist of i) activation of polymer, ii) conjugation, and iii)
blocking of residual active groups.
[0139] In the following a number of suitable polymer activation
methods will be described shortly. However, it is to be understood
that also other methods may be used.
[0140] Coupling polymeric molecules to the free acid groups of
polypeptides may be performed with the aid of diimide and for
example amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr.
Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992,
"Chemistry of Protein Conjugation and Crosslinking", CRC
Press).
[0141] Coupling polymeric molecules to hydroxy groups are generally
very difficult as it must be performed in water. Usually hydrolysis
predominates over reaction with hydroxyl groups.
[0142] Coupling polymeric molecules to free sulfhydryl groups can
be reached with special groups like maleimido or the ortho-pyridyl
disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow
et al.) has a preference for sulfhydryl groups but is not as
selective as the other mentioned.
[0143] Accessible arginine residues in the polypeptide chain may be
targeted by groups comprising two vicinal carbonyl groups.
[0144] Techniques involving coupling electrophilically activated
PEGs to the amino groups of lysines may also be useful. Many of the
usual leaving groups for alcohols give rise to an amine linkage.
For instance, alkyl sulfonates, such as tresylates (Nilsson et al.,
1984, Methods in Enzymology, 104, Jacoby, W. B., Ed., Academic
Press: Orlando, 56-66; Nilsson et al., 1987, Methods in Enzymology,
135; Mosbach, K., Ed.; Academic Press: Orlando, 65-79; Scouten et
al., 1987, Methods in Enzymology, 135, Mosbach, K., Ed., Academic
Press: Orlando, 1987, 79-84; Crossland et al., 1971, J. Amr. Chem.
Soc., 93, 4217-4219), mesylates (Harris, (1985), supra; Harris et
al., 1984, J. Polym. Sci. Polym. Chem. Ed., 22, 341-352), aryl
sulfonates like tosylates, and para-nitrobenzene sulfonates can be
used.
[0145] Organic sulfonyl chlorides, e.g. tresyl chloride,
effectively converts hydroxy groups in a number of polymers, e.g.
PEG, into good leaving groups (sulfonates) that, when reacted with
nucleophiles like amino groups in polypeptides allow stable
linkages to be formed between polymer and polypeptide. In addition
to high conjugation yields, the reaction conditions are in general
mild (neutral or slightly alkaline pH, to avoid denaturation and
little or no disruption of activity), and satisfy the
non-destructive requirements to the polypeptide.
[0146] Tosylate is more reactive than the mesylate but also more
unstable decomposing into PEG, dioxane, and sulfonic acid
(Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also
been used for creating amine bonds but are much less reactive than
the above mentioned groups.
[0147] Converting PEG into a chloroformate with phosgene gives rise
to carbamate linkages to lysines. This theme can be played in many
variants substituting the chlorine with N-hydroxy succinimide (U.S.
Pat. No. 5,122,614, (1992); Zalipsky et al., 1992, Biotechnol.
Appl. Biochem., 15, 100-114; Monfardini et al., 1995, Bioconjugate
Chem., 6, 62-69, with imidazole (Allen et al., 1991, Carbohydr.
Res., 213, 309-319), with para-nitrophenol, DMAP (EP 632 082, 1993,
Looze, Y.) etc. The derivatives are usually made by reacting the
chloroformate with the desired leaving group. All these groups give
rise to carbamate linkages to the peptide.
[0148] Furthermore, isocyanates and isothiocyanates may be employed
yielding ureas and thioureas, respectively.
[0149] Amides may be obtained from PEG acids using the same leaving
groups as mentioned above and cyclic imide thrones (U.S. Pat. No.
5,349,001 (1994), Greenwald et al.). The reactivity of these
compounds is very high but may make the hydrolysis to fast.
[0150] PEG succinate made from reaction with succinic anhydride can
also be used. The hereby comprised ester group make the conjugate
much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, 1992,
Zalipsky). This group may be activated with N-hydroxy
succinimide.
[0151] Furthermore, a special linker can be introduced. The oldest
being cyanuric chloride (Abuchowski et al., 1977, J. Biol. Chem.,
252, 3578-3581; U.S. Pat. No. 4,179,337, 1979, Davis et al.; Shafer
et al., 1986, J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.
[0152] Coupling of PEG to an aromatic amine followed by
diazotization yields a very reactive diazonium salt which in situ
can be reacted with a peptide. An amide linkage may also be
obtained by reacting an azlactone derivative of PEG (U.S. Pat. No.
5,321,095, 1994, Greenwald, R. B.) thus introducing an additional
amide linkage.
[0153] As some peptides do not comprise many lysines, it may be
advantageous to attach more than one PEG to the same lysine. This
can be done e.g. by the use of 1,3-diamino-2-propanol.
[0154] PEGs may also be attached to the amino-groups of the enzyme
with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine
residues may also be used as the backbone.
[0155] The coupling technique used in the examples is the
N-succinimidyl carbonate conjugation technique described in WO
90/13590 (Enzon).
[0156] Method for Preparing Improved Conjugates
[0157] It is also an object of the invention to provide a method
for preparing improved polypeptide-polymer conjugates comprising
the steps of:
[0158] a) identifying amino acid residues located on the surface of
the 3D structure of the parent polypeptide in question,
[0159] b) selecting target amino acid residues on the surface of
said 3D structure of said parent polypeptide to be mutated,
[0160] c) i) substituting or inserting one or more amino acid
residues selected in step b) with an amino acid residue having a
suitable attachment group, and/or ii) substituting or deleting one
or more amino acid residues selected in step b) at or close to the
functional site(s),
[0161] d) coupling polymeric molecules to the mutated
polypeptide.
[0162] Step a) Identifying Amino Acid Residues Located on the
Surface of the Parent Polypeptide
[0163] 3-Dimensional Structure (3D-Structure)
[0164] To perform the method of the invention a 3-dimensional
structure of the parent polypeptide in question is required. This
structure may for example be an X-ray structure, an NMR structure
or a model-built structure. The Brookhaven Databank is a source of
X-ray- and NMR-structures.
[0165] A model-built structure may be produced by the person
skilled in the art if one or more 3D-structure(s) exist(s) of
homologous polypeptide(s) sharing at least 30% sequence identity
with the polypeptide in question. Several software packages exist
which may be employed to construct a model structure. One example
is the Homology 95.0 package from Biosym.
[0166] Typical actions required for the construction of a model
structure are: alignment of homologous sequences for which
3D-structures exist, definition of Structurally Conserved Regions
(SCRs), assignment of coordinates to SCRs, search for structural
fragments/loops in structure databases to replace Variable Regions,
assignment of coordinates to these regions, and structural
refinement by energy minimization. Regions containing large inserts
(>3 residues) relative to the known 3D-structures are known to
be quite difficult to model, and structural predictions must be
considered with care.
[0167] Having obtained the 3D-structure of the polypeptide in
question, or a model of the structure based on homology to known
structures, this structure serves as an essential prerequisite for
the fulfillment of the method described below.
[0168] Step b) Selection of Target Amino Acid Residues for
Mutation
[0169] Target amino acid residues to be mutated are according to
the invention selected in order to obtain additional or fewer
attachment groups, such as free amino groups (--NH.sub.2) or free
carboxylic acid groups (--COOH), on the surface of the polypeptide
and/or to obtain a more complete and broadly spread shielding of
the epitope(s) on the surface of the polypeptide.
[0170] Conservative Substitution
[0171] It is preferred to make conservative substitutions in the
polypeptide, as conservative substitutions secure that the impact
of the mutation on the polypeptide structure is limited.
[0172] In the case of providing additional amino groups this may be
done by substitution of arginine to lysine, which are both
positively charged, but only the lysine having a free amino group
suitable as an attachment group.
[0173] In the case of providing additional carboxylic acid groups
the conservative substitution may for instance be an aspargine to
aspartic acid or glutamine to glutamic acid substitution. These
residues resemble each other in size and shape, except from the
carboxylic groups being present on the acidic residues.
[0174] In the case of providing fewer attachment groups, e.g. at or
close to the active site, a lysine may be substituted with an
arginine, and so on.
[0175] Which amino acids to substitute depends in principle on the
coupling chemistry to be applied.
[0176] Non-Conservative Substitution
[0177] The mutation may also be on target amino acid residues which
are less/non-conservative. Such mutation is suitable for obtaining
a more complete and broadly spread shielding of the polypeptide
surface than can be obtained by the conservative substitutions.
[0178] The method of the invention is first described in general
terms, and subsequently using specific examples.
[0179] Note the use of the following terms:
[0180] Attachment_residue: residue(s) which can bind polymeric
molecules, e.g. lysines (amino group) or aspartic/glutamic acids
(carboxylic groups). N- or C-terminal amino/carboxylic groups are
to be included where relevant.
[0181] Mutation_residue: residue(s) which is to be mutated, e.g.
arginine or aspargine/glutamine.
[0182] Essential_catalytic_residues: residues which are known to be
essential for catalytic function, e.g. the catalytic triad in
serine proteases.
[0183] Solvent_exposed_residues: These are defined as residues
which are at least 5% exposed according to the BIOSYM/INSIGHT
algorithm found in the module Homology 95.0. The sequence of
commands is as follows:
Homology=>ProStat=>Access_Surf=>Solv_Radius 1.4; Heavy
atoms only; Radii source VdW; Output: Fractional Area; Polarity
source: Default. The file filename_area.tab is produced. Note: For
this program to function properly all water molecules must first be
removed from the structure.
[0184] It looks for example like:
2 # PD498FINALMODEL # residue area TRP_1 136.275711 SER_2 88.188095
PRO_3 15.458788 ASN_4 95.322319 ASP_5 4.903404 PRO_6 68.096909
TYR_7 93.333252 TYR_8 31.791576 SER_9 95.983139 . . . continued
[0185] 1. Identification of residues which are more than 10
Angstroms away from the closest attachment_residue, and which are
located at least 8 Angstroms away from
essential_catalytic_residues. This residue subset is called REST,
and is the primary region for conservative mutation_residue to
attachment_residue substitutions.
[0186] 2. Identification of residues which are located in a 0-5
Angstroms shell around subset REST, but at least 8 Angstroms away
from essential_catalytic_residues. This residue subset is called
SUB5B. This is a secondary region for conservative mutation_residue
to attachment_residue substitutions, as a ligand bound to an
attachment_residue in SUB5B will extend into the REST region and
potentially prevent epitope recognition.
[0187] 3. Identification of solvent_exposed mutation_residues in
REST and SUB5B as potential mutation sites for introduction of
attachment_residues.
[0188] 4. Use BIOSYM/INSIGHT's Biopolymer module and replace
residues identified under action 3.
[0189] 5. Repeat 1-2 above producing the subset RESTx. This subset
includes residues which are more than 10 Angstroms away from the
nearest attachment_residue, and which are located at least 8
Angstroms away from essential catalytic residues.
[0190] 6. Identify solvent_exposed residues in RESTx. These are
potential sites for less/non-conservative mutations to introduce
atttachment_residues.
[0191] Step c) Substituting, Inserting or Deleting Amino Acid
Residues
[0192] The mutation(s) performed in step c) may be performed by
standard techniques well known in the art, such as site-directed
mutagenesis (see, e.g., Sambrook et al., 1989, Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, N.Y.
[0193] A general description of nucleotide substitution can be
found in e.g. Ford et al., 1991, Protein Expression and
Purification, 2, 95-107.
[0194] Step d) Coupling Polymeric Molecules to the Modified Parent
Enzyme
[0195] Polypeptide-polymer conjugates of the invention may be
prepared by any coupling method known in the art including the
above mentioned techniques.
[0196] Coupling of Polymeric Molecules to the Polypeptide in
Question
[0197] If the polymeric molecules to be conjugated with the
polypeptide are not active it must be activated by the use of a
suitable method. The polymeric molecules may be coupled to the
polypeptide through a linker. Suitable linkers are well known to
the skilled person.
[0198] Methods and chemistry for activation of polymeric molecules
as well as for conjugation of polypeptides are intensively
described in the literature. Commonly used methods for activation
of insoluble polymers include activation of functional groups with
cyanogen bromide, periodate, glutaraldehyde, biepoxides,
epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,
trichlorotriazine etc. (see R. F. Taylor, 1991, "Protein
Immobilisation. Fundamentals and Applications", Marcel Dekker,
N.Y.; S. S. Wong, 1992, "Chemistry of Protein Conjugation and
Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al., 1993,
"Immobilized Affinity Ligand Techniques", Academic Press, N.Y.).
Some of the methods concern activation of insoluble polymers but
are also applicable to activation of soluble polymers e.g.
periodate, trichlorotriazine, sulfonylhalides, divinylsulfone,
carbodiimide etc. The functional groups being amino, hydroxyl,
thiol, carboxyl, aldehyde or sulfydryl on the polymer and the
chosen attachment group on the protein must be considered in
choosing the activation and conjugation chemistry which normally
consists of i) activation of polymer, ii) conjugation, and iii)
blocking of residual active groups.
[0199] In the following a number of suitable polymer activation
methods will be described shortly. However, it is to be understood
that also other methods may be used.
[0200] Coupling polymeric molecules to the free acid groups of
enzymes can be performed with the aid of diimide and for example
amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr. Chem.
Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992,
"Chemistry of Protein Conjugation and Crosslinking", CRC
Press).
[0201] Coupling polymeric molecules to hydroxy groups are generally
very difficult as it must be performed in water. Usually hydrolysis
predominates over reaction with hydroxyl groups.
[0202] Coupling polymeric molecules to free sulfhydryl groups can
be reached wih special groups like maleimido or the ortho-pyridyl
disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135 (1995), Snow
et al.) has a preference for sulfhydryl groups but is not as
selective as the other mentioned.
[0203] Accessible arginine residues in the polypeptide chain may be
targeted by groups comprising two vicinal carbonyl groups.
[0204] Techniques involving coupling electrophilically activated
PEGs to the amino groups of lysines are also useful. Many of the
usual leaving groups for alcohols give rise to an amine linkage.
For instance, alkyl sulfonates, such as tresylates (Nilsson et al.,
1984, Methods in Enzymology, 104, Jacoby, W. B., Ed., Academic
Press: Orlando, 56-66; Nilsson et al., (1987), Methods in
Enzymology, 135; Mosbach, K., Ed.; Academic Press: Orlando, 65-79;
Scouten et al., 1987, Methods in Enzymology, 135, Mosbach, K., Ed.,
Academic Press: Orlando, 1987; 79-84; Crossland et al., 1971, J.
Amr. Chem. Soc., 1971, 93, 4217-4219), mesylates (Harris, 1985,
supra; Harris et al., 1984, J. Polym. Sci. Polym. Chem. Ed., 22,
341-352), aryl sulfonates like tosylates, and para-nitrobenzene
sulfonates can be used.
[0205] Organic sulfonyl chlorides, e.g. tresyl chloride,
effectively converts hydroxy groups in a number of polymers, e.g.
PEG, into good leaving groups (sulfonates) that, when reacted with
nucleophiles like amino groups in polypeptides allow stable
linkages to be formed between polymer and polypeptide. In addition
to high conjugation yields, the reaction conditions are in general
mild (neutral or slightly alkaline pH, to avoid denaturation and
little or no disruption of activity), and satisfy the
non-destructive requirements to the polypeptide.
[0206] Tosylate is more reactive than the mesylate but also more
unstable decomposing into PEG, dioxane, and sulfonic acid
(Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also
been used for creating amine bonds but are much less reactive than
the above mentioned groups.
[0207] Converting PEG into a chloroformate with phosgene gives rise
to carbamate linkages to lysines. This theme can be played in many
variants substituting the chlorine with N-hydroxy succinimide (U.S.
Pat. No. 5,122,614 (1992); Zalipsky et al., 1992, Biotechnol. Appl.
Biochem., 15, 100-114; Monfardini et al., 1995, Bioconjugate Chem.,
6, 62-69, with imidazole (Allen et al., 1991, Carbohydr. Res., 213,
309-319), with para-nitrophenol, DMAP (EP 632 082, 1993, Looze, Y.)
etc. The derivatives are usually made by reacting the chloroformate
with the desired leaving group. All these groups give rise to
carbamate linkages to the peptide.
[0208] Furthermore, isocyanates and isothiocyanates may be employed
yielding ureas and thioureas, respectively.
[0209] Amides may be obtained from PEG acids using the same leaving
groups as mentioned above and cyclic imide thrones (U.S. Pat. No.
5,349,001 (1994), Greenwald et al.). The reactivity of these
compounds is very high but may make the hydrolysis to fast.
[0210] PEG succinate made from reaction with succinic anhydride can
also be used. The hereby comprised ester group makes the conjugate
much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614,
(1992), Zalipsky). This group may be activated with N-hydroxy
succinimide.
[0211] Furthermore, a special linker can be introduced. The oldest
being cyanuric chloride (Abuchowski et al., 1977, J. Biol. Chem.,
252, 3578-3581; U.S. Pat. No. 4,179,337, 1979, Davis et al.; Shafer
et al., 1986, J. Polym. Sci. Polym. Chem. Ed., 24, 375-378).
[0212] Coupling of PEG to an aromatic amine followed by
diazotization yields a very reactive diazonium salt which in situ
can be reacted with a peptide. An amide linkage may also be
obtained by reacting an azlactone derivative of PEG (U.S. Pat. No.
5,321,095, (1994), Greenwald, R. B.) thus introducing an additional
amide linkage.
[0213] As some peptides do not comprise many lysines, it may be
advantageous to attach more than one PEG to the same lysine. This
can be done e.g. by the use of 1,3-diamino-2-propanol.
[0214] PEGs may also be attached to the amino-groups of the enzyme
with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine
residues may also be used as the backbone.
[0215] Addition of Attachment Groups
[0216] Specific Examples of PD498 Variant-SPEG Conjugates
[0217] A specific example of a protease is the parent PD498 (WO
93/24623 and SEQ ID NO: 2). The parent PD498 has a molecular weight
of 29 kDa.
[0218] Lysine and arginine residues are located as follows:
3 Distance from the active site Arginine Lysine 0-5 Angstroms 1
5-10 Angstroms 10-15 Angstroms 5 6 15-20 Angstroms 2 3 20-25
Angstroms 1 3 Total 9 12
[0219] The inventors examined which parent PD498 sites on the
surface may be suitable for introducing additional attachment
groups.
[0220] A. Suitable conservative arginine to lysine substitutions in
parent PD498 may be any of R51K, R62K, R121K, R169K, R250K, R28K,
R190K.
[0221] B. Suitable non-conservative substitutions in parent PD498
may be any of P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K,
N65K, G87K, 188K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K,
Y221K, G262K.
[0222] As there are no lysine residues at or close to the active
site there is no need for removing any attachment group.
[0223] PD498 variant-SPEG conjugates may be prepared using any of
the above mentioned PD498 variants as the starting material by any
conjugation technique known in the art for coupling polymeric
molecules to amino groups on the enzyme. A specific example is
described below.
[0224] Removal of Attachment Groups
[0225] Specific Examples of BPN' Variant-SPEG Conjugates
[0226] A specific example of a protease having an attachment group
in the active site is BPN' which has 11 attachment groups (plus an
N-terminal amino group): BPN' has a molecular weight of 28 kDa.
[0227] Lysine and arginine residues are located as follows:
4 Distance from the active site Arginine Lysine 0-5 Angstroms 1
5-10 Angstroms 10-15 Angstroms 1 4 15-20 Angstroms 1 4 20-25
Angstroms 2 Total 2 11
[0228] The lysine residue located within 0-5 Angstroms of the
active site can according to the invention advantageously be
removed. Specifically this may be done by a K94R substitution.
[0229] BPN' variant-SPEG conjugates may be prepared using the above
mentioned BPN' variant as the starting material by any conjugation
technique known in the art for coupling polymeric molecules to
amino groups on the enzyme.
[0230] Addition and Removal of Attachment Groups
[0231] Specific Example of SAVINASE.RTM.-SPEG Conjugates
[0232] As described in Example 2 parent SAVINASE.RTM. (von der
Osten et al., 1993, Journal of Biotechnology, 28, 55+ and SEQ ID
NO: 3) may according to the invention have added a number of amino
attachment groups to the surface and removed an amino attachment
group close to the active site.
[0233] Any of the following substitutions in SAVINASE.RTM. are
sites for mutagenesis: R10K, R19K, R45K, R145K, R170K, R186K and
R247K.
[0234] The substitution K94R is identified as a mutation suitable
for preventing attachment of polymers close to active site.
[0235] SAVINASE.RTM. variant-SPEG conjugates may be prepared using
any of the above mentioned SAVINASE.RTM. variants as the starting
material by any conjugation technique known in the art for coupling
polymeric molecules to amino groups on the enzyme.
[0236] Addition of Attachment Groups
[0237] Specific Examples of Humicola lanuginosa Lipase
Variants-SPEG Conjugates
[0238] Specific examples of lipase variants with reduced
immunogenicity using the parent Huminocal lanuginosa DSM 4109
lipase (see SEQ ID NO: 6) as the backbone for substitutions are
listed below.
[0239] The parent unmodified Humicola lanuginosa lipase has 8
attachment groups including the N-terminal NH.sub.2 group and a
molecular weight of about 29 kDa.
[0240] Suitable conservative arginine to lysine substitutions in
the parent lipase may be any of R133K, R139K, R160K, R179K, R209K,
R118K and R125K.
[0241] Suitable non-conservative substitutions in the parent lipase
may be any of: A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K,
E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K,
N92K, L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K,
P250K, F262K.
[0242] Further suitable non-conservative substitution in the
Humicola lanuginosa lipase include: E87K or D254K.
[0243] Lipase variant-SPEG conjugates may be prepared using any of
the above mentioned lipase variants as the starting material by any
conjugation technique known in the art for coupling polymeric
molecules to amino groups on the enzyme. A specific example is
described below.
[0244] In Example 12 below it is shown that a conjugate of the
Humicola lanuginosa lipase variant with E87K.sup.+ D254K
substitutions coupled to S-PEG 15,000 has reduced immunogenic
response in Balb/C mice in comparison to the corresponding parent
unmodified enzyme.
[0245] Immunogenicity and Allergenicity
[0246] "Immunogenicity" is a broader term than "antigenicity" and
"allergenicity", and expresses the immune system's response to the
presence of foreign substances. Said foreign substances are called
immunogens, antigens and allergens depending of the type of immune
response they elicit.
[0247] An "immunogen" may be defined as a substance which, when
introduced into circulatory system of animals and humans, is
capable of stimulating an immunologic response resulting in
formation of immunoglobulin.
[0248] The term "antigen" refers to substances which by themselves
are capable of generating antibodies when recognized as a non-self
molecule.
[0249] Further, an "allergen" may be defined as an antigen which
may give rise to allergic sensitization or an allergic response by
IgE antibodies (in humans, and molecules with comparable effects in
animals).
[0250] Assessment of Immunogenicity
[0251] Assessment of the immunogenicity may be made by injecting
animal subcutaneously to enter the immunogen into the circulation
system and comparing the response with the response of the
corresponding parent polypeptide.
[0252] The "circulatory system" of the body of humans and animals
means, in the context of the present invention, the system which
mainly consists of the heart and blood vessels. The heart delivers
the necessary energy for maintaining blood circulation in the
vascular system. The circulation system functions as the organism's
transportation system, when the blood transports O.sub.2,
nutritious matter, hormones, and other substances of importance for
the cell regulation into the tissue. Further the blood removes
CO.sub.2 from the tissue to the lungs and residual substances to
e.g. the kidneys. Furthermore, the blood is of importance for the
temperature regulation and the defense mechanisms of the body,
which include the immune system.
[0253] A number of in vitro animal models exist for assessment of
the immunogenic potential of polypeptides. Some of these models
give a suitable basis for hazard assessment in man. Suitable models
include a mice model.
[0254] This model seeks to identify the immunogenic response in the
form of the IgG response in Balb/C mice being injected
subcutaneously with modified and unmodified polypeptides.
[0255] Also other animal models can be used for assessment of the
immunogenic potential.
[0256] A polypeptide having "reduced immunogenicity" according to
the invention indicates that the amount of produced antibodies,
e.g. immunoglobulin in humans, and molecules with comparable
effects in specific animals, which can lead to an immune response,
is significantly decreased, when introduced into the circulatory
system, in comparison to the corresponding parent polypeptide.
[0257] For Balb/C mice the IgG response gives a good indication of
the immunigenic potential of polypeptides.
[0258] Assessment of Allergenicity
[0259] Assessment of allergenicity may be made by inhalation tests,
comparing the effect of intratracheally (into the trachea)
administrated parent enzymes with the corresponding modified
enzymes according to the invention.
[0260] A number of in vivo animal models exist for assessment of
the allegenicity of enzymes. Some of these models give a suitable
basis for hazard assessment in man. Suitable models include a
guinea pig model and a mouse model. These models seek to identify
respiratory allergens as a function of elicitation reactions
induced in previously sensitized animals. According to these models
the alleged allergens are introduced intratracheally into the
animals.
[0261] A suitable strain of guinea pigs, the Dunkin Hartley strain,
does not as humans, produce IgE antibodies in connection with the
allergic response. However, they produce another type of antibody
the IgG1A and IgG1B (see e.g. Prent.o slashed., ATLA, 19, 8-14,
1991), which are responsible for their allergenic response to
inhaled polypeptides including enzymes. Therefore, when using the
Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B
is a measure of the allergenicity level.
[0262] The Balb/C mice strain is suitable for intratracheal
exposure. Balb/C mice produce IgE as the allergic response.
[0263] More details on assessing respiratory allergens in guinea
pigs and mice are described by Kimber et al., 1996, Fundamental and
Applied Toxicology, 33, 1-10.
[0264] Other animals such as rats, rabbits etc. may also be used
for comparable studies.
[0265] Composition
[0266] The invention relates to a composition comprising a
polypeptide-polymer conjugate of the invention.
[0267] The composition may be a pharmaceutical or industrial
composition.
[0268] The composition may further comprise other polypeptides,
proteins or enzymes and/or ingredients normally used in e.g.
detergents, including soap bars, household articles, agrochemicals,
personal care products, including skin care compositions, cleaning
compositions for e.g. contact lenses, oral and dermal
pharmaceuticals, composition use for treating textiles,
compositions used for manufacturing food, e.g. baking, and feed
etc.
[0269] Use of the Polypeptide-Polymer Conjugate
[0270] The invention also relates to the use of the method of the
invention for reducing the immune response of polypeptides.
[0271] It is also an object of the invention to use the
polypeptide-polymer conjugate of the invention to reduce the
allergenicity of industrial products, such as detergents, such as
laundry, dish wash and hard surface cleaning detergents, and food
or feed products.
[0272] Material and Methods
[0273] Materials
[0274] Enzymes:
[0275] PD498: Protease of subtilisin type shown in WO 93/24623. The
sequence of PD498 is shown in SEQ ID NOS: 1 and 2.
[0276] SAVINASE.RTM. (Available from Novo Nordisk A/S)
[0277] Humicola lanuginosa lipase: Available from Novo Nordisk as
LIPOLASE.RTM. and is further described in EP 305,216. The DNA and
protein sequence is shown in SEQ ID NOS: 5 and 6, respectively.
[0278] Strains:
[0279] B. subtilis 309 and 147 are variants of Bacillus lentus,
deposited with the NCIB and accorded the accession numbers NCIB
10309 and 10147, and described in U.S. Pat. No. 3,723,250
incorporated by reference herein.
[0280] E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J.
Mol. Biol. 138 179-207), was made r.sup.-,m.sup.+ by conventional
methods and is also described in US Patent Application Serial No.
039,298.
[0281] Vectors:
[0282] pPD498: E. coli-B. subtilis shuttle vector (described in
U.S. Pat. No. 5,621,089 under section 6.2.1.6) containing the
wild-type gene encoding for PD498 protease (SEQ ID NO: 2). The same
vector is used for mutagenesis in E. coli as well as for expression
in B. subtilis.
[0283] General Molecular Biology Methods:
[0284] Unless otherwise mentioned the DNA manipulations and
transformations were performed using standard methods of molecular
biology (Sambrook et al., 1989, Molecular cloning: A laboratory
Manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel,
F. M. et al. (eds.) "Current protocols in Molecular Biology". John
Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)
"Molecular Biological Methods for Bacillus". John Wiley and Sons,
1990).
[0285] Enzymes for DNA manipulations were used according to the
specifications of the suppliers.
[0286] Materials, Chemicals and Solutions:
[0287] Horse Radish Peroxidase labeled anti-rat-Ig (Dako, DK, P162,
# 031; dilution 1:1000).
[0288] Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).
[0289] Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).
[0290] Biotin-labeled mouse anti-rat IgG1 monoclonal antibody
(Zymed 03-9140; dilution 1:1000)
[0291] Biotin-labeled rat anti-mouse IgG1 monoclonal antibody
(Serotec MCA336B; dilution 1:1000)
[0292] Streptavidin-horse radish peroxidase (Kirkeg.ang.rd &
Perry 14-30-00; dilution 1:1000).
[0293] CovaLink NH.sub.2 plates (Nunc, Cat# 459439)
[0294] Cyanuric chloride (Aldrich)
[0295] Acetone (Merck)
[0296] Rat anti-Mouse IgG1, biotin (SeroTec, Cat# MCA336B)
[0297] Streptavidin, peroxidase (KPL)
[0298] Ortho-Phenylene-diamine (OPD) (Kem-en-Tec)
[0299] H.sub.2O.sub.2, 30% (Merck)
[0300] Tween 20 (Merck)
[0301] Skim Milk powder (Difco)
[0302] H.sub.2SO.sub.4 (Merck).
[0303] Buffers and Solutions:
5 Carbonate buffer (0.1 M, pH 10 (1 liter)) Na.sub.2CO.sub.3 10.60
g PBS (pH 7.2 (1 liter)) NaCl 8.00 g KCl 0.20 g K.sub.2HPO.sub.4
1.04 g KH.sub.2PO.sub.4 0.32 g Washing buffer PBS, 0.05% (v/v)
Tween 20 Blocking buffer PBS, 2% (wt/v) Skim Milk powder Dilution
buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder
Citrate buffer (0.1 M, pH 5.0-5.2 (1 liter)) NaCitrate 20.60 g
Citric acid 6.30 g
[0304] Activation of CovaLink Plates:
[0305] Make a fresh stock solution of 10 mg cyanuric chloride per
ml acetone.
[0306] Just before use, dilute the cyanuric chloride stock solution
into PBS, while stirring, to a final concentration of 1 mg/ml.
[0307] Add 100 ml of the dilution to each well of the CovaLink
NH.sub.2 plates, and incubate for 5 minutes at room
temperature.
[0308] Wash 3 times with PBS.
[0309] Dry the freshly prepared activated plates at 50.degree. C.
for 30 minutes.
[0310] Immediately seal each plate with sealing tape.
[0311] Preactivated plates can be stored at room temperature for 3
weeks when kept in a plastic bag.
[0312] Sodium Borate, borax (Sigma)
[0313] 3,3-Dimethyl glutaric acid (Sigma)
[0314] CaCl.sub.2 (Sigma)
[0315] Tresyl chloride (2,2,2-triflouroethansulfonyl chloride)
(Fluka)
[0316] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
(Fluka)
[0317] N-Hydroxy succinimide (Fluka art. 56480))
[0318] Phosgene (Fluka art. 79380)
[0319] Lactose (Merck 7656)
[0320] PMSF (phenyl methyl sulfonyl flouride) from Sigma
[0321]
Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide
(Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.
[0322] Coloring Substrate:
[0323] OPD: o-phenylene-diamine, (Kementec cat no. 4260).
[0324] Test Animals:
[0325] Dunkin Hartley guinea pigs (from Charles River, DE)
[0326] Female Balb/C mice (about 20 grams) purchased from
Bomholdtgaard, Ry, Denmark.
[0327] Equipment:
[0328] XCEL II (Novex)
[0329] ELISA reader (UVmax, Molecular Devices)
[0330] HPLC (Waters)
[0331] PFLC (Pharmacia)
[0332] Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.
[0333] SLT: Fotometer from SLT LabInstruments
[0334] Size-exclusion chromatograph (Spherogel TSK-G2000 SW).
[0335] Size-exclusion chromatograph (Superdex 200, Pharmacia,
SW)
[0336] Amicon Cell.
[0337] Enzymes for DNA Manipulations
[0338] Unless otherwise mentioned all enzymes for DNA
manipulations, such as e.g. restriction endonucleases, ligases
etc., are obtained from New England Biolabs, Inc.
[0339] Methods
[0340] ELISA Procedure for Determination of IgG.sub.1 Positive
Guinea Pigs
[0341] ELISA microtiter plates are coated with rabbit anti-PD498
1:8000 in carbonate buffer and incubated overnight at 4.degree. C.
The next day the plates are blocked with 2% BSA for 1 hour and
washed 3 times with PBS Tween 20.
[0342] 1 microgram/ml PD498 is added to the plates and incubated
for 1 hour, then washed 3 times with PBS Tween 20.
[0343] All guinea pig sera samples and controls are applied to the
ELISA plates with 2 microliters sera and 98 microliters PBS,
incubated for 1 hour and washed 3 times with PBS Tween 20.
[0344] Then goat anti-guinea pig IgG.sub.1 (1:4000 in PBS buffer
(Nordic Immunology, 44-682)) is applied to the plates, incubated
for 1 hour and washed with PBS tween 20.
[0345] Alkaline phosphatase marked rabbit anti-goat 1:8000 (Sigma
A4187) is applied and incubated for 1 hour, washed 2 times in PBS
Tween20 and 1 time with diethanol amine buffer.
[0346] The marked alkaline phosphatase is developed using
p-nitrophenyl phosphate for 30 minutes at 37.degree. C. or until
appropriate color has developed.
[0347] The reaction is stopped using stop medium
(K.sub.2HPO.sub.4/HaH.sub- .3 buffer comprising EDTA (pH 10)) and
read at OD 405/650 using an ELISA reader.
[0348] Double blinds are included on all ELISA plates.
[0349] Positive and negative sera values are calculated as the
average blind values added 2 times the standard deviation. This
gives an accuracy of 95%.
[0350] Determination of the Molecule Weight
[0351] Electrophoretic separation of proteins was performed by
standard methods using 4-20% gradient SDS poly acrylamide gels
(Novex). Proteins were detected by silver staining. The molecule
weight was measured relative to the mobility of Mark-12@ wide range
molecule weight standards from Novex.
[0352] Protease Activity
[0353] Analysis with Suc-Ala-Ala-Pro-Phe-pNa:
[0354] Proteases cleave the bond between the peptide and
p-nitroaniline to give a visible yellow color absorbing at 405
nm.
[0355] Buffer: e.g. Britton and Robinson buffer pH 8.3.
[0356] Substrate: 100 mg suc-AAPF-pNa is dissolved into 1 ml
dimethyl sulfoxide (DMSO). 100 microliters of this is diluted into
10 ml with Britton and Robinson buffer.
[0357] The substrate and protease solution is mixed and the
absorbance is monitored at 405 nm as a function of time and
ABS.sub.405 nm/min. The temperature should be controlled
(20-50.degree. C. depending on protease). This is a measure of the
protease activity in the sample.
[0358] Proteolytic Activity
[0359] In the context of this invention proteolytic activity is
expressed in Kilo NOVO Protease Units (KNPU). The activity is
determined relatively to an enzyme standard (SAVINASE.RTM.), and
the determination is based on the digestion of a dimethyl casein
(DMC) solution by the proteolytic enzyme at standard conditions,
i.e. 50.degree. C., pH 8.3, 9 min. reaction time, 3 min. measuring
time. A folder AF 220/1 is available upon request to Novo Nordisk
A/S, Denmark, which folder is hereby included by reference.
[0360] A Glycine Unit (GU) is defined as the proteolytic enzyme
activity which, under standard conditions, during a 15-minute
incubation at 40.degree. C., with N-acetyl casein as substrate,
produces an amount of NH.sub.2-group equivalent to 1 mmole of
glycine.
[0361] Enzyme activity can also be measured using the PNA assay,
according to reaction with the soluble substrate
succinyl-alanine-alanine-proline-p- henyl-alanine-para-nitrophenol,
which is described in Rothgeb, T. M., Goodlander, B. D., Garrison,
P. H., and Smith, L. A., 1988 Journal of American Oil Chemists
Society.
[0362] Fermentation of PD498 Variants
[0363] Fermentation of PD498 variants in B. subtilis are performed
at 30.degree. C. on a rotary shaking table (300 r.p.m.) in 500 ml
baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days.
In order to make an e.g. 2 liter broth 20 Erlenmeyer flasks are
fermented simultaneously.
[0364] Media:
6 BPX: Composition (per liter) Potato starch 100 g Ground barley 50
g Soybean flour 20 g Na.sub.2HPO.sub.4 .times. 12 H.sub.2O 9 g
Pluronic 0.1 g Sodium caseinate 10 g
[0365] The starch in the medium is liquefied with alpha-amylase and
the medium is sterilized by heating at 120.degree. C. for 45
minutes. After sterilization the pH of the medium is adjusted to 9
by addition of NaHCO.sub.3 to 0.1 M.
[0366] Purification of PD498 Variants
[0367] Approximately 1.6 liters of PD498 variant fermentation broth
are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The
supernatants are adjusted to pH 7.0 using 10% acetic acid and
filtered on Seitz Supra S100 filter plates.
[0368] The filtrates are concentrated to approximately 400 ml using
an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge.
The UF concentrate is centrifuged and filtered prior to absorption
at room temperature on a Bacitracin affinity column at pH 7. The
PD498 variant is eluted from the Bacitracin column at room
temperature using 25% 2-propanol and 1 M sodium chloride in a
buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid
and 0.002 M calcium chloride adjusted to pH 7.
[0369] The fractions with protease activity from the Bacitracin
purification step are combined and applied to a 750 ml Sephadex G25
column (5 cm diameter) equilibrated with a buffer containing 0.01
dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium
chloride adjusted to pH 6.0.
[0370] Fractions with proteolytic activity from the Sephadex G25
column are combined and applied to a 150 ml CM Sepharose CL 6B
cation exchange column (5 cm diameter) equilibrated with a buffer
containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and
0.002 M calcium chloride adjusted to pH 6.0.
[0371] The protease is eluted using a linear gradient of 0-0.5 M
sodium chloride in 1 liter of the same buffer.
[0372] Protease containing fractions from the CM Sepharose column
are combined and filtered through a 2 micron filter.
[0373] Balb/C Mice IgG ELISA Procedure:
[0374] The antigen is diluted to 1 mg/ml in carbonate buffer.
[0375] 100 ml is added to each well.
[0376] The plates are coated overnight at 4.degree. C.
[0377] Unspecific adsorption is blocked by incubating each well for
1 hour at room temperature with 200 ml blocking buffer.
[0378] The plates are washed 3.times. with 300 ml washing
buffer.
[0379] Unknown mouse sera are diluted in dilution buffer, typically
10.times., 20.times. and 40.times., or higher.
[0380] 100 ml is added to each well.
[0381] Incubation is for 1 hour at room temperature.
[0382] Unbound material is removed by washing 3.times. with washing
buffer.
[0383] The anti-Mouse IgG1 antibody is diluted 2000.times. in
dilution buffer.
[0384] 100 ml is added to each well.
[0385] Incubation is for 1 hour at room temperature.
[0386] Unbound material is removed by washing 3.times. with washing
buffer.
[0387] Streptavidine is diluted 1000.times. in dilution buffer.
[0388] 100 ml is added to each well.
[0389] Incubation is for 1 hour at room temperature.
[0390] Unbound material is removed by washing 3.times. with 300 ml
washing buffer.
[0391] OPD (0.6 mg/ml) and H.sub.2O.sub.2 (0.4 ml/ml) is dissolved
in citrate buffer.
[0392] 100 ml is added to each well.
[0393] Incubation is for 10 minutes at room temperature.
[0394] The reaction is stopped by adding 100 ml
H.sub.2SO.sub.4.
[0395] The plates are read at 492 nm with 620 nm as reference.
[0396] Immunization of Mice
[0397] Balb/C mice (20 grams) are immunized 10 times (intervals of
14 days) by subcutaneous injection of the modified or unmodified
polypeptide in question, respectively by standard procedures known
in art.
EXAMPLES
Example 1
[0398] Suitable Substitutions in PD498 for Addition of Amino
Attachment Groups (--NH.sub.2)
[0399] The 3D structure of parent PD498 was modeled as described
above based on 59% sequence identity with Thermitase.RTM.
(2tec.pdb).
[0400] The sequence of PD498 is SEQ ID NO: 2. PD498 residue
numbering is used, 1-280.
[0401] The commands performed in Insight (BIOSYM) are shown in the
command files makeKzone.bcl and makeKzone2.bcl below:
[0402] Conservative Substitutions:
[0403] makeKzone.bcl
[0404] 1 Delete Subset *
[0405] 2 Color Molecule Atoms * Specified Specification
55,0,255
[0406] 3 Zone Subset LYS :lys:NZ Static monomer/residue 10
Color_Subset 255,255,0
[0407] 4 Zone Subset NTERM :1:N Static monomer/residue 10
Color_Subset 255,255,0
[0408] 5 #NOTE: editnextline ACTSITE residues according to the
protein
[0409] 6 Zone Subset ACTSITE :39,72,226 Static monomer/residue 8
Color_Subset 255,255,0
[0410] 7 Combine Subset ALLZONE Union LYS NTERM
[0411] 8 Combine Subset ALLZONE Union ALLZONE ACTSITE
[0412] 9 #NOTE: editnextline object name according to the
protein
[0413] 10 Combine Subset REST Difference PD498FINALMODEL
ALLZONE
[0414] 11 List Subset REST Atom Output_File restatom.list
[0415] 12 List Subset REST monomer/residue Output_File
restmole.list
[0416] 13 Color Molecule Atoms ACTSITE Specified Specification
255,0,0
[0417] 14 List Subset ACTSITE Atom Output_File actsiteatom.list
[0418] 15 List Subset ACTSITE monomer/residue Output_File
actsitemole.list
[0419] 16 #
[0420] 17 Zone Subset REST5A REST Static Monomer/Residue
5-Color_Subset
[0421] 18 Combine Subset SUB5A Difference REST5A ACTSITE
[0422] 19 Combine Subset SUB5B Difference SUB5A REST
[0423] 20 Color Molecule Atoms SUB5B Specified Specification
255,255,255
[0424] 21 List Subset SUB5B Atom Output_File sub5batom.list
[0425] 22 List Subset SUB5B monomer/residue Output_File
sub5bmole.list
[0426] 23 #Now identify sites for lys->arg substitutions and
continue with makezone2.bcl
[0427] 24 #Use grep command to identify ARG in restatom.list,
sub5batom.list & accsiteatom.list.
[0428] Comments:
[0429] Lines 1-8: The subset ALLZONE is defined as those residues
which are either within 10 Angstroms of the free amino groups on
lysines or the N-terminal, or within 8 Angstroms of the catalytic
triad residues 39, 72 and 226.
[0430] Line 10: The subset REST is defined as those residues not
included in ALLZONE.
[0431] Lines 17-20: Subset SUB5B is defined as those residues in a
5 Angstroms shell around REST, excluding residues within 8
Angstroms of the catalytic residues.
[0432] Line 23-24: REST contains Arg62 and Arg169, SUB5B contains
Arg51, Arg121, and Arg250. ACTSITE contains Arg103, but position
103 is within 8 Angstroms from essential_catalytic_residues, and
thus not relevant.
[0433] The color codes are: (255,0,255)=magenta, (255,255,0)
yellow, (255,0,0) red, and (255, 255, 255)=white.
[0434] The substitutions R51K, R62K, R121K, R169K and R250K are
identified in parent PD498 as suitable sites for mutagenesis. The
residues are substituted below in section 2, and further analysis
done:
[0435] Non-Conservative Substitutions:
[0436] makeKzone2.bcl
[0437] 1 #sourcefile makezone2.bcl Claus von der Osten 961128
[0438] 2 #
[0439] 3 #having scanned lists (grep arg command) and identified
sites for lys->arg substitutions
[0440] 4 #NOTE: editnextline object name according to protein
[0441] 5 Copy Object -To_Clipboard -Displace PD498FINALMODEL
newmodel 6 Biopolymer
[0442] 7 #NOTE: editnextline object name according to protein
[0443] 8 Blank Object On PD498FINALMODEL
[0444] 9 #NOTE: editnextlines with lys->arg positions Replace
Residue newmodel:51 lys L
[0445] 11 Replace Residue newmodel:62 lys L
[0446] 12 Replace Residue newmodel:121 lys L
[0447] 13 Replace Residue newmodel:169 lys L
[0448] 14 Replace Residue newmodel:250 lys L
[0449] 15 #
[0450] 16 #Now repeat analysis done prior to arg->lys, now
including introduced lysines
[0451] 17 Color Molecule Atoms newmodel Specified Specification
255,0,255
[0452] 18 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue
10 Color_Subset 255,255,0
[0453] 19 Zone Subset NTERMx newmodel:l:N Static monomer/residue 10
Color_Subset 255,255,0
[0454] 20 #NOTE: editnextline ACTSITEx residues according to the
protein
[0455] 21 Zone Subset ACTSITEx newmodel:39,72,226 Static
monomer/residue 8 Color_Subset 255,255,0
[0456] 22 Combine Subset ALLZONEx Union LYSx NTERMx
[0457] 23 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
[0458] 24 Combine Subset RESTx Difference newmodel ALLZONEx List
Subset RESTx Atom Output_File restxatom.list
[0459] 26 List Subset RESTx monomer/residue Output_File
restxmole.list
[0460] 27 #
[0461] 28 Color Molecule Atoms ACTSITEx Specified Specification
255,0,0
[0462] 29 List Subset ACTSITEx Atom Output_File
actsitexatom.list
[0463] 30 List Subset ACTSITEx monomer/residue Output_File
actsitexmole.list
[0464] 31 #
[0465] 32 #read restxatom.list or restxmole.list to identify sites
for (not_arg)->lys subst. if needed.
[0466] Comments:
[0467] Lines 1-15: Solvent exposed arginines in subsets REST and
SUB5B are replaced by lysines. Solvent accessibilities are
recalculated following arginine replacement.
[0468] Lines 16-23: The subset ALLZONEx is defined as those
residues which are either within 10 Angstroms of the free amino
groups on lysines (after replacement) or the N-terminal, or within
8 Angstroms of the catalytic triad residues 39, 72 and 226.
[0469] Line 24-26: The subset RESTx is defined as those residues
not included in ALLZONEx, i.e. residues which are still potential
epitope contributors. Of the residues in RESTx, the following are
>5% exposed (see lists below): 6-7,9-12, 43-45, 65, 87-88, 209,
211, 216-221, 262.
[0470] The following mutations are proposed in parent PD498: P6K,
Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, 188K,
N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K.
[0471] Relevant Data for Example 1:
[0472] Solvent Accessibility Data for PD498MODEL:
7 # PD498MODEL Fri Nov 29 10:24:48 MET 1996 # residue area TRP_1
136.275711 SER_2 88.188095 PRO_3 15.458788 ASN_4 95.322319 ASP_5
4.903404 PRO_6 68.096909 TYR_7 93.333252 TYR_8 31.791576 SER_9
95.983139 ALA_10 77.983536 TYR_11 150.704727 GLN_12 26.983349
TYR_13 44.328232 GLY_14 3.200084 PRO_15 2.149547 GLN_16 61.385445
ASN_17 37.776707 THR_18 1.237873 SER_19 41.031750 THR_20 4.321402
PRO_21 16.658991 ALA_22 42.107288 ALA_23 0.000000 TRP_24 3.713619
ASP_25 82.645493 VAL_26 74.397812 THR_27 14.950654 ARG_28
110.606209 GLY_29 0.242063 SER_30 57.225292 SER_31 86.986198 THR_32
1.928865 GLN_33 42.008949 THR_34 0.502189 VAL_35 0.268693 ALA_36
0.000000 VAL_37 5.255383 LEU_38 1.550332 ASP_39 3.585718 SER_40
2.475746 GLY_41 4.329043 VAL_42 1.704864 ASP_43 25.889742 TYR_44
89.194855 ASN_45 109.981819 HIS_46 0.268693 PRO_47 66.580925 ASP_48
0.000000 LEU_49 0.770882 ALA_50 49.618046 ARG_51 218.751709 LYS_52
18.808538 VAL_53 39.937984 ILE_54 98.478104 LYS_55 103.612228
GLY_56 17.199390 TYR_57 67.719147 ASP_58 0.000000 PHE_59 40.291119
ILE_60 50.151962 ASP_61 70.078888 ARG_62 166.777557 ASP_63
35.892376 ASN_64 120.641953 ASN_65 64.982895 PRO_66 6.986028 MET_67
58.504269 ASP_68 28.668840 LEU_69 104.467468 ASN_70 78.460953
GLY_71 5.615932 HIS_72 43.158905 GLY_73 0.268693 THR_74 0.000000
HIS_75 0.484127 VAL_76 1.880854 ALA_77 0.000000 GLY_78 0.933982
THR_79 9.589676 VAL_80 0.000000 ALA_81 0.000000 ALA_82 0.000000
ASP_83 46.244987 THR_84 27.783333 ASN_85 75.924225 ASN_86 44.813908
GLY_87 50.453152 ILE_88 74.428070 GLY_89 4.115077 VAL_90 6.717335
ALA_91 2.872341 GLY_92 0.233495 MET_93 5.876057 ALA_94 0.000000
PRO_95 17.682203 ASP_96 83.431740 THR_97 1.506567 LYS_98 72.674973
ILE_99 4.251006 LEU_100 6.717335 ALA_101 0.806080 VAL_102 1.426676
ARG_103 2.662697 VAL_104 2.171855 LEU_105 18.808538 ASP_106
52.167435 ALA_107 52.905663 ASN_108 115.871315 GLY_109 30.943356
SER_110 57.933651 GLY_111 50.705326 SER_112 56.383320 LEU_113
71.312195 ASP_114 110.410919 SER_115 13.910152 ILE_116 22.570246
ALA_117 5.642561 SER_118 29.313131 GLY_119 0.000000 ILE_120
1.343467 ARG_121 118.391129 TYR_122 44.203033 ALA_123 0.000000
ALA_124 7.974043 ASP_125 83.851639 GLN_126 64.311974 GLY_127
36.812618 ALA_128 4.705107 LYS_129 90.886139 VAL_130 1.039576
LEU_131 2.149547 ASN_132 4.315227 LEU_133 1.880854 SER_134 3.563334
LEU_135 26.371397 GLY_136 59.151070 CYS_137 63.333755 GLU_138
111.553314 CYS_139 83.591461 ASN_140 80.757843 SER_141 25.899158
THR_142 99.889725 THR_143 73.323814 LEU_144 5.589301 LYS_145
94.708755 SER_146 72.636993 ALA_147 9.235920 VAL_148 1.612160
ASP_149 57.431465 TYR_150 106.352493 ALA_151 0.268693 TRP_152
43.133667 ASN_153 112.864975 LYS_154 110.009468 GLY_155 33.352180
ALA_156 3.493014 VAL_157 1.048144 VAL_158 2.043953 VAL_159 0.000000
ALA_160 0.537387 ALA_161 10.872165 ALA_162 7.823834 GLY_163
12.064573 ASN_164 81.183388 ASP_165 64.495300 ASN_166 83.457443
VAL_167 68.516815 SER_168 78.799652 ARG_169 116.937134 THR_170
57.275074 PHE_171 51.416462 GLN_172 18.934589 PRO_173 1.880854
ALA_174 6.522357 SER_175 26.184139 TYR_176 21.425076 PRO_177
85.613541 ASN_178 34.700817 ALA_179 0.268693 ILE_180 1.074774
ALA_181 3.761708 VAL_182 0.000000 GLY_183 2.149547 ALA_184 0.951118
ILE_185 0.806080 ASP_186 30.022263 SER_187 72.518509 ASN_188
117.128021 ASP_189 47.601345 ARG_190 150.050873 LYS_191 64.822807
ALA_192 2.686934 SER_193 96.223808 PHE_194 51.482613 SER_195
1.400973 ASN_196 4.148808 TYR_197 80.937309 GLY_198 10.747736
THR_199 93.221252 TRP_200 169.943604 VAL_201 15.280325 ASP_202
12.141763 VAL_203 0.268693 THR_204 3.409728 ALA_205 0.000000
PRO_206 0.000000 GLY_207 0.000000 VAL_208 37.137192 ASN_209
78.286270 ILE_210 9.404268 ALA_211 25.938599 SER_212 5.037172
THR_213 0.000000 VAL_214 22.301552 PRO_215 45.251030 ASN_216
131.014160 ASN_217 88.383461 GLY_218 21.226780 TYR_219 88.907570
SER_220 39.966541 TYR_221 166.037018 MET_222 50.951096 SER_223
54.435001 GLY_224 1.880854 THR_225 1.634468 SER_226 17.432346
MET_227 7.233279 ALA_228 0.000000 SER_229 0.000000 PRO_230 0.268693
HIS_231 2.680759 VAL_232 0.000000 ALA_233 0.000000 GLY_234 1.074774
LEU_235 11.500556 ALA_236 0.000000 ALA_237 0.000000 LEU_238
1.612160 LEU_239 0.000000 ALA_240 10.648088 SER_241 39.138004
GLN_242 71.056175 GLY_243 66.487144 LYS_244 43.256012 ASN_245
80.728127 ASN_246 34.859673 VAL_247 84.145645 GLN_248 51.819775
ILE_249 8.598188 ARG_250 35.055809 GLN_251 71.928093 ALA_252
0.000000 ILE_253 4.845899 GLU_254 13.344438 GLN_255 81.705254
THR_256 9.836061 ALA_257 2.810513 ASP_258 44.656136 LYS_259
113.071686 ILE_260 32.089527 SER_261 91.590103 GLY_262 26.450439
THR_263 38.308762 GLY_264 46.870056 THR_265 88.551804 ASN_266
34.698349 PHE_267 7.756911 LYS_268 103.212852 TYR_269 37.638382
GLY_270 0.000000 LYS_271 11.376978 ILE_272 2.885231 ASN_273
19.195255 SER_274 2.651736 ASN_275 38.177547 LYS_276 84.549576
ALA_277 1.074774 VAL_278 4.775503 ARG_279 162.693054 TYR_280
96.572929 CA_281 0.000000 CA_282 0.000000 CA_283 8.803203 Subset
REST: restmole.list Subset REST: PD498FINALMODEL: 6-7, 9-12, 43-46,
61-63, 65, 87-89, 111-114, 117-118, 131, PD498FINALMODEL: 137-139,
158-159, 169-171, 173-174, 180-181, 209, 211, PD498FINALMODEL:
216-221, 232-233, 262, E282H restatom.list Subset REST:
PD498FINALMODEL: PRO 6: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
TYR 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
PD498FINALMODEL: SER 9: N, CA, C, O, CB, OG PD498FINALMODEL: ALA
10: N, CA, C, O, CB PD498FINALMODEL: TYR 11: N, CA, C, O, CB, CG,
CD1, CD2, CE1, CE2, CZ, OH PD498FINALMODEL: GLN 12: N, CA, C, O,
CB, CG, CD, OE1, NE2 PD498FINALMODEL: ASP 43: N, CA, C, O, CB, CG,
OD1, OD2 PD498FINALMODEL: TYR 44: N, CA, C, O, CB, CG, CD1, CD2,
CE1, CE2, CZ, OH PD498FINALMODEL: ASN 45: N, CA, C, O, CB, CG, OD1,
ND2 PD498FINALMODEL: HIS 46: N, CA, C, O, CB, CG, ND1, CD2, CE1,
NE2 PD498FINALMODEL: ASP 61: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: ARG 62: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
PD498FINALMODEL: ASP 63: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: ASN 65: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: GLY 87: N, CA, C, O PD498FINALMODEL: ILE 88: N,
CA, C, O, CB, CG1, CG2, GD1 PD498FINALMODEL: GLY 89: N, CA, C, O
PD498FINALMODEL: GLY 111: N, CA, C, O PD498FINALMODEL: SER 112: N,
CA, C, O, CB, OG PD498FINALMODEL: LEU 113: N, CA, C, O, CB, CG,
CD1, CD2 PD498FINALMODEL: ASP 114: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: ALA 117: N, CA, C, O, CB PD498FINALMODEL: SER 118:
N, CA, C, O, CB, OG PD498FINALMODEL: LEU 131: N, CA, C, O, CB, CG,
CD1, CD2 PD498FINALMODEL: CYS 137: N, CA, C, O, CB, SG
PD498FINALMODEL: GLU 138: N, CA, C, O, CB, CG, CD, OE1, OE2
PD498FINALMODEL: CYS 139: N, CA, C, O, CB, SG PD498FINALMODEL: VAL
158: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL: VAL 159: N, CA, C,
O, CB, CG1, CG2 PD498FINALMODEL: ARG 169: N, CA, C, O, CB, CG, CD,
NE, CZ, NH1, NH2 PD498FINALMODEL: THR 170: N, CA, C, O, CB, OG1,
CG2 PD498FINALMODEL: PHE 171: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ PD498FINALMODEL: PRO 173: N, CA, CD, C, O, CB, CG
PD498FINALMODEL: ALA 174: N, CA, C, O, CB PD498FINALMODEL: ILE 180:
N, CA, C, O, CB, CG1, CG2, CD1 PD498FINALMODEL: ALA 181: N, CA, C,
O, CB PD498FINALMODEL: ASN 209: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: ALA 211: N, CA, C, O, CB PD498FINALMODEL: ASN 216:
N, CA, C, O, CB, CG, OD1, ND2 PD498FINALMODEL: ASN 217: N, CA, C,
O, CB, CG, OD1, ND2 PD498FINALMODEL: GLY 218: N, CA, C, O
PD498FINALMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: SER 220: N, CA, C, O, CB, OG
PD498FINALMODEL: TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: VAL 232: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: ALA 233: N, CA, C, O, CB PD498FINALMODEL: GLY 262:
N, CA, C, O PD498FINALMODEL: CA E282H: CA Subset SUB5B:
sub5bmole.list Subset SUB5B: PD498FINALMODEL: 4-5, 8, 13-16, 34-35,
47-51, 53, 64, 83, 85-86, 90-91, 120-124, PD498FINALMODEL: 128-130,
140-141, 143-144, 147-148, 151-152, 156-157, PD498FINALMODEL: 165,
167-168, 172, 175-176, 178-179, 196, 200-205, 208, PD498FINALMODEL:
234-237, 250, 253-254, 260-261, 263-267, 272, E281H,
PD498FINALMODEL: E283H sub5batom.list Subset SUB5B:
PD498FINALMODEL: ASN 4: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: ASP 5: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: TYR 8: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: TYR 13: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ, OH PD498FINALMODEL: GLY 14: N, CA, C, O PD498FINALMODEL:
PRO 15: N, CA, CD, C, O, CB, CG PD498FINALMODEL: GLN 16: N, CA, C,
O, CB, CG, CD, OE1, NE2 PD498FINALMODEL: THR 34: N, CA, C, O, CB,
OG1, CG2 PD498FINALMODEL: VAL 35: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: PRO 47: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
ASP 48: N, CA, C, O, CB, CG, OD1, OD2 PD498FINALMODEL: LEU 49: N,
CA, C, O, CB, CG, CD1, CD2 PD498FINALMODEL: ALA 50: N, CA, C, O, CB
PD498FINALMODEL: ARG 51: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
PD498FINALMODEL: VAL 53: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL:
ASN 64: N, CA, C, O, CB, CG, OD1, ND2 PD498FINALMODEL: ASP 83: N,
CA, C, O, CB, CG, OD1, OD2 PD498FINALMODEL: ASN 85: N, CA, C, O,
CB, CG, OD1, ND2 PD498FINALMODEL: ASN 86: N, CA, C, O, CB, CG, OD1,
ND2 PD498FINALMODEL: VAL 90: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: ALA 91: N, CA, C, O, CB PD498FINALMODEL: ILE 120:
N, CA, C, O, CB, CG1, CG2, CD1 PD498FINALMODEL: ARG 121: N, CA, C,
O, CB, CG, CD, NE, CZ, NH1, NH2 PD498FINALMODEL: TYR 122: N, CA, C,
O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH PD498FINALMODEL: ALA 123: N,
CA, C, O, CB PD498FINALMODEL: ALA 124: N, CA, C, O, CB
PD498FINALMODEL: ALA 128: N, CA, C, O, CB PD498FINALMODEL: LYS 129:
N, CA, C, O, CB, CG, CD, CE, NZ PD498FINALMODEL: VAL 130: N, CA, C,
O, CB, CG1, CG2 PD498FINALMODEL: ASN 140: N, CA, C, O, CB, CG, OD1,
ND2 PD498FINALMODEL: SER 141: N, CA, C, O, CB, OG PD498FINALMODEL:
THR 143: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL: LEU 144: N, CA,
C, O, CB, CG, CD1, CD2 PD498FINALMODEL: ALA 147: N, CA, C, O, CB
PD498FINALMODEL: VAL 148: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: ALA 151: N, CA, C, O, CB PD498FINALMODEL: TRP 52:
N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
PD498FINALMODEL: ALA 156: N, CA, C, O, CB PD498FINALMODEL: VAL 157:
N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL: ASP 165: N, CA, C, O,
CB, CG, OD1, OD2 PD498FINALMODEL: VAL 167: N, CA, C, O, CB, CG1,
CG2 PD498FINALMODEL: SER 168: N, CA, C, O, CB, OG PD498FINALMODEL:
GLN 172: N, CA, C, O, CB, CG, CD, OE1, NE2 PD498FINALMODEL: SER
175: N, CA, C, O, CB, OG PD498FINALMODEL: TYR 176: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ, OH PD498FINALMODEL: ASN 178: N, CA, C,
O, CB, CG, OD1, ND2 PD498FINALMODEL: ALA 179: N, CA, C, O, CB
PD498FINALMODEL: ASN 196: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: TRP 200: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2,
CE3, CZ2, CZ3, CH2 PD498FINALMODEL: VAL 201: N, CA, C, O, CB, CG1,
CG2 PD498FINALMODEL: ASP 202: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: VAL 203: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: THR 204: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: ALA 205: N, CA, C, O, CB PD498FINALMODEL: VAL 208:
N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL: GLY 234: N, CA, C, O
PD498FINALMODEL: LEU 235: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ALA 236: N, CA, C, O, CB PD498FINALMODEL: ALA 237:
N, CA, C, O, CB PD498FINALMODEL: ARG 250: N, CA, C, O, CB, CG, CD,
NE, CZ, NH1, NH2 PD498FINALMODEL: ILE 253: N, CA, C, O, CB, CG1,
CG2, GD1 PD498FINALMODEL: GLU 254: N, CA, C, O, CB, CG, CD, OE1,
OE2 PD498FINALMODEL: ILE 260: N, CA, C, O, CB, CG1, CG2, GD1
PD498FINALMODEL: SER 261: N, CA, C, O, CB, OG PD498FINALMODEL: THR
263: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL: GLY 264: N, CA, C,
O PD498FINALMODEL: THR 265: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: ASN 266: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: PHE 267: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ PD498FINALMODEL: ILE 272: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: CA E281H: CA PD498FINALMODEL: CA E283H: NA Subset
ACTSITE: actsitemole.list Subset ACTSITE: PD498FINALMODEL: 36-42,
57-60, 66-80, 100-110, 115-116, 119, 132-136, 160-164,
PD498FINALMODEL: 182-184, 194, 206-207, 210, 212-215, 222-231
actsiteatom.list Subset ACTSITE: PD498FINALMODEL: ALA 36: N, CA, C,
O, CB PD498FINALMODEL: VAL 37: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: LEU 38: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ASP 39: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: SER 40: N, CA, C, O, CB, OG PD498FINALMODEL: GLY
41: N, CA, C, O PD498FINALMODEL: VAL 42: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: TYR 57: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: ASP 58: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: PHE 59: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ PD498FINALMODEL: ILE 60: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: PRO 66: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
MET 67: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: ASP 68: N, CA,
C, O, CB, CG, OD1, OD2 PD498FINALMODEL: LEU 69: N, CA, C, O, CB,
CG, CD1, CD2 PD498FINALMODEL: ASN 70: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: GLY 71: N, CA, C, O PD498FINALMODEL: HIS 72: N,
CA, C, O, CB, CG, ND1, CD2, CE1, NE2 PD498FINALMODEL: GLY 73: N,
CA, C, O PD498FINALMODEL: THR 74: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: HIS 75: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
PD498FINALMODEL: VAL 76: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL:
ALA 77: N, CA, C, O, CB PD498FINALMODEL: GLY 78: N, CA, C, O
PD498FINALMODEL: THR 79: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL:
VAL 80: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL: LEU 100: N, CA,
C, O, CB, CG, CD1, CD2 PD498FINALMODEL: ALA 101: N, CA, C, O, CB
PD498FINALMODEL: VAL 102: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: ARG 103: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
PD498FINALMODEL: VAL 104: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: LEU 105: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ASP 106: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: ALA 107: N, CA, C, O, CB PD498FINALMODEL: ASN 108:
N, CA, C, O, CB, CG, OD1, ND2 PD498FINALMODEL: GLY 109: N, CA, C, O
PD498FINALMODEL: SER 110: N, CA, C, O, CB, OG PD498FINALMODEL: SER
115: N, CA, C, O, CB, OG PD498FINALMODEL: ILE 116: N, CA, C, O, CB,
CG1, CG2, CD1 PD498FINALMODEL: GLY 119: N, CA, C, O
PD498FINALMODEL: ASN 132: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: LEU 133: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: SER 134: N, CA, C, O, CB, OG
PD498FINALMODEL: LEU 135: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: GLY 136: N, CA, C, O PD498FINALMODEL: ALA 160: N,
CA, C, O, CB PD498FINALMODEL: ALA 161: N, CA, C, O, CB
PD498FINALMODEL: ALA 162: N, CA, C, O, CB PD498FINALMODEL: GLY 163:
N, CA, C, O PD498FINALMODEL: ASN 164: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: VAL 182: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: GLY 183: N, CA, C, O PD498FINALMODEL: ALA 184: N,
CA, C, O, CB PD498FINALMODEL: PHE 194: N, CA, C, O, CB, CG, CD1,
CD2, CE1, CE2, CZ PD498FINALMODEL: PRO 206: N, CA, CD, C, O, CB, CG
PD498FINALMODEL: GLY 207: N, CA, C, O PD498FINALMODEL: ILE 210: N,
CA, C, O, CB, CG1, CG2, CD1 PD498FINALMODEL: SER 212: N, CA, C, O,
CB, OG PD498FINALMODEL: THR 213: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: VAL 214: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: PRO 215: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
MET 222: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: SER 223: N,
CA, C, O, CB, OG PD498FINALMODEL: GLY 224: N, CA, C, O
PD498FINALMODEL: THR 225: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: SER 226: N, CA, C, O, CB, OG PD498FINALMODEL: MET
227: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: ALA 228: N, CA,
C, O, CB PD498FINALMODEL: SER 229: N, CA, C, O, CB, OG
PD498FINALMODEL: PRO 230: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
HIS 231: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2 Subset RESTx:
restxmole.list Subset RESTX: NEWMODEL: 6-7, 9-12, 43-46, 65, 87-89,
131, 173, 209, 211, 216-221, 232-233, NEWMODEL: 262, E282H
restxatom.list Subset RESTX: NEWMODEL: PRO 6: N, CA, CD, C, O, CB,
CG NEWMODEL: TYR 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
NEWMODEL: SER 9: N, CA, C, O, CB, OG NEWMODEL: ALA 10: N, CA, C, O,
CB NEWMODEL: TYR 11: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ,
OH NEWMODEL: GLN 12: N, CA, C, O, CB, CG, CD, OE1, NE2 NEWMODEL:
ASP 43: N, CA, C, O, CB, CG, OD1, OD2 NEWMODEL: TYR 44: N, CA, C,
O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH NEWMODEL: ASN 45: N, CA, C,
O, CB, CG, OD1, ND2 NEWMODEL: HIS 46: N, CA, C, O, CB, CG, ND1,
CD2, CE1, NE2 NEWMODEL: ASN 65: N, CA, C, O, CB, CG, OD1, ND2
NEWMODEL: GLY 87: N, CA, C, O NEWMODEL: ILE 88: N, CA, C, O, CB,
CG1, CG2, CD1 NEWMODEL: GLY 89: N, CA, C, O NEWMODEL: LEU 131: N,
CA, C, O, CB, CG, CD1, CD2 NEWMODEL: PRO 173: N, CA, CD, C, O, CB,
CG NEWMODEL: ASN 209: N, CA, C, O, CB, CG, OD1, ND2 NEWMODEL: ALA
211: N, CA, C, O, CB NEWMODEL: ASN 216: N, CA, C, O, CB, CG, OD1,
ND2 NEWMODEL: ASN 217: N, CA, C, O, CB, CG, OD1, ND2 NEWMODEL: GLY
218: N, CA, C, O NEWMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2,
CE1, CE2, CZ, OH NEWMODEL: SER 220: N, CA, C, O, CB, OG NEWMODEL:
TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH NEWMODEL:
VAL 232: N, CA, C, O, CB, CG1, CG2 NEWMODEL: ALA 233: N, CA, C, O,
CB NEWMODEL: GLY 262: N, CA, C, O NEWMODEL: CA E282H: CA
Example 2
[0473] Suitable Substitutions in SAVINASE.RTM. for Addition of
Amino Attachment Groups (--NH.sub.2)
[0474] The known X-ray structure of SAVINASE.RTM. was used to find
where suitable amino attachment groups may is added (Betzel et al,
1992, J. Mol. Biol., 223, 427-445).
[0475] The 3D structure of SAVINASE.RTM. is available in the
Brookhaven Databank as lsvn.pbd. A related subtilisin is available
as 1st3.pdb.
[0476] The sequence of SAVINASE.RTM. is shown in SEQ ID NO: 3. The
sequence numbering used is that of subtilisin BPN', SAVINASE.RTM.
having deletions relative to BPN' at positions 36, 56, 158-159 and
163-164. The active site residues (functional site) are D32, H64
and S221.
[0477] The commands performed in Insight (BIOSYM) are shown in the
command files makeKzone.bcl and makeKzone2.bcl below:
[0478] Conservative Substitutions:
[0479] makeKzone.bcl
[0480] Delete Subset *
[0481] Color Molecule Atoms * Specified Specification 255,0,255
[0482] Zone Subset LYS :lys:NZ Static monomer/residue 10
Color_Subset 255,255,0
[0483] Zone Subset NTERM :e1:N Static monomer/residue 10
Color_Subset 255,255,0
[0484] #NOTE: editnextline ACTSITE residues according to the
protein
[0485] Zone Subset ACTSITE :e32,e64,e221 Static monomer/residue 8
Color_Subset 255,255,0
[0486] Combine Subset ALLZONE Union LYS NTERM
[0487] Combine Subset ALLZONE Union ALLZONE ACTSITE
[0488] #NOTE: editnextline object name according to the protein
[0489] Combine Subset REST Difference SAVI8 ALLZONE
[0490] List Subset REST Atom Output_File restatom.list
[0491] List Subset REST monomer/residue Output_File
restmole.list
[0492] Color Molecule Atoms ACTSITE Specified Specification
255,0,0
[0493] List Subset ACTSITE Atom Output_File actsiteatom.list
[0494] List Subset ACTSITE monomer/residue Output_File
actsitemole.list
[0495] #
[0496] Zone Subset REST5A REST Static Monomer/Residue
5-Color_Subset
[0497] Combine Subset SUB5A Difference REST5A ACTSITE
[0498] Combine Subset SUB5B Difference SUB5A REST
[0499] Color Molecule Atoms SUB5B Specified Specification
255,255,255
[0500] List Subset SUB5B Atom Output_File sub5batom.list
[0501] List Subset SUB5B monomer/residue Output_File
subsbmole.list
[0502] #Now identify sites for lys->arg substitutions and
continue with makezone2.bcl
[0503] #Use grep command to identify ARG in restatom.list,
sub5batom.list & accsiteatom.list.
[0504] Comments:
[0505] In this case of SAVINASE.RTM. REST contains the arginines
Arg10, Arg170 and Arg 186, and SUB5B contains Arg19, Arg45, Arg145
and Arg247.
[0506] These residues are all solvent exposed. The substitutions
R10K, R19K, R45K, R145K, R170K, R186K and R247K are identified in
SAVINASE.RTM. as sites for mutagenesis within the scope of this
invention. The residues are substituted below in section 2, and
further analysis done. The subset ACTSITE contains Lys94.
[0507] The substitution K94R is a mutation removing lysine as
attachment group close to the active site.
[0508] Non-Conservative Substitutions:
[0509] makeKzone2.bcl
[0510] #sourcefile makezone2.bcl Claus von der Osten 961128
[0511] #
[0512] #having scanned lists (grep arg command) and identified
sites for lys->arg substitutions
[0513] #NOTE: editnextline object name according to protein
[0514] Copy Object -To_Clipboard -Displace SAVI8 newmodel
[0515] Biopolymer
[0516] #NOTE: editnextline object name according to protein
[0517] Blank Object On SAVI8
[0518] #NOTE: editnextlines with lys->arg positions
[0519] Replace Residue newmodel:e10 lys L
[0520] Replace Residue newmodel:e170 lys L
[0521] Replace Residue newmodel:e186 lys L
[0522] Replace Residue newmodel:e19 lys L
[0523] Replace Residue newmodel:e45 lys L
[0524] Replace Residue newmodel:e145 lys L
[0525] Replace Residue newmodel:e241 lys L
[0526] #
[0527] #Now repeat analysis done prior to arg->lys, now
including introduced lysines
[0528] Color Molecule Atoms newmodel Specified Specification
255,0,255
[0529] Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10
Color_Subset 255,255,0
[0530] Zone Subset NTERMx newmodel:e1:N Static monomer/residue 10
Color_Subset 255,255,0
[0531] #NOTE: editnextline ACTSITEx residues according to the
protein
[0532] Zone Subset ACTSITEx newmodel:e32,e64,e221 Static
monomer/residue 8 Color_Subset 255,255,0
[0533] Combine Subset ALLZONEx Union LYSx NTERMx
[0534] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
[0535] Combine Subset RESTx Difference newmodel ALLZONEx
[0536] List Subset RESTx Atom Output_File restxatom.list
[0537] List Subset RESTx monomer/residue Output_File
restxmole.list
[0538] #
[0539] Color Molecule Atoms ACTSITEx Specified Specification
255,0,0
[0540] List Subset ACTSITEx Atom Output_File actsitexatom.list
[0541] List Subset ACTSITEx monomer/residue Output_File
actsitexmole.list
[0542] #
[0543] #read restxatom.list or restxmole.list to identify sites for
(not_arg)->lys subst. if needed.
[0544] Comments:
[0545] Of the residues in RESTx, the following are >5% exposed
(see lists below): 5, 14, 22, 38-40, 42, 75-76, 82, 86, 103-105,
108, 133-135, 137, 140, 173, 204, 206, 211-213, 215-216, 269. The
following mutations are proposed in SAVINASE.RTM.: P5K, P14K, T22K,
T38K, H39K, P40K, L42K, L75K, N76K, L82K, P86K, S103K, V104K,
S105K, A108K, A133K, T134K, L135K, Q137K, N140K, N173K, N204K,
Q206K, G211K, S212K, T213K, A215K, S216K, N269K.
[0546] Relevant Data for Example 2:
[0547] Solvent Accessibility Data for SAVINASE.RTM.:
8 # SAVI8NOH2O Fri Nov 29 13: 32: 07 MET 1996 # residue area ALA_1
118.362808 GLN_2 49.422764 SER_3 61.982887 VAL_4 71.620255 PRO_5
21.737535 TRP_6 58.718731 GLY_7 4.328117 ILE_8 6.664074 SER_9
60.175900 ARG_10 70.928963 VAL_11 2.686934 GLN_12 72.839996 ALA_13
0.000000 PRO_14 52.308453 ALA_15 38.300892 ALA_16 0.000000 HIS_17
41.826324 ASN_18 136.376602 ARG_19 105.678642 GLY_20 48.231510
LEU_21 17.196377 THR_22 36.781742 GLY_23 0.000000 SER_24 64.151276
GLY_25 50.269905 VAL_26 4.030401 LYS_27 54.239555 VAL_28 0.000000
ALA_29 0.000000 VAL_30 3.572827 LEU_31 0.233495 ASP_32 1.074774
THR_33 1.973557 GLY_34 3.638052 ILE_35 8.044439 SER_36 8.514903
THR_37 122.598907 HIS_38 18.834011 PRO_39 76.570526 ASP_40 0.000000
LEU_41 19.684013 ASN_42 88.870216 ILE_43 56.117710 ARG_44
110.647194 GLY_45 26.935413 GLY_46 35.515778 ALA_47 21.495472
SER_48 34.876190 PHE_49 52.647541 VAL_50 23.364208 PRO_51
110.408752 GLY_52 80.282906 GLU_53 43.033707 PRO_54 124.444336
SER_55 60.284889 THR_56 47.103241 GLN_57 120.803505 ASP_58
12.784743 GLY_59 61.742443 ASN_60 56.760231 GLY_61 1.576962 HIS_62
38.590118 GLY_63 0.000000 THR_64 0.537387 HIS_65 0.968253 VAL_66
1.612160 ALA_67 0.000000 GLY_68 2.801945 THR_69 9.074596 ILE_70
0.000000 ALA_71 4.577205 ALA_72 0.000000 LEU_73 47.290039 ASN_74
102.187248 ASN_75 60.210400 SER_76 84.614494 ILE_77 66.098572
GLY_78 17.979534 VAL_79 5.642561 LEU_80 13.025185 GLY_81 0.000000
VAL_82 0.268693 ALA_83 0.000000 PRO_84 18.193810 SER_85 56.839039
ALA_86 13.075745 GLU_87 37.011765 LEU_88 2.149547 TYR_89 30.633518
ALA_90 1.343467 VAL_91 0.779450 LYS_92 5.862781 VAL_93 0.466991
LEU_94 10.747736 GLY_95 8.707102 ALA_96 41.414677 SER_97 96.066040
GLY_98 33.374485 SER_99 67.664116 GLY_100 35.571117 SER_101
54.096992 VAL_102 52.695324 SER_103 62.929684 SER_104 8.683097
ILE_105 15.852910 ALA_106 14.509443 GLN_107 94.463066 GLY_108
0.000000 LEU_109 0.537387 GLU_110 63.227707 TRP_111 55.500740
ALA_112 0.502189 GLY_113 11.908267 ASN_114 107.208527 ASN_115
78.811234 GLY_116 41.453194 MET_117 9.634291 HIS_118 54.022118
VAL_119 5.105174 ALA_120 0.268693 ASN_121 0.233495 LEU_122 0.537387
SER_123 4.004620 LEU_124 21.927265 GLY_125 55.952454 SER_126
40.241180 PRO_127 107.409439 SER_128 57.988609 PRO_129 85.021118
SER_130 20.460915 ALA_131 57.404362 THR_132 74.438805 LEU_133
12.091203 GLU_134 73.382019 GLN_135 114.870010 ALA_136 2.122917
VAL_137 1.074774 ASN_138 55.622704 SER_139 29.174965 ALA_140
0.268693 THR_141 27.962946 SER_142 87.263145 ARG_143 88.201218
GLY_144 38.477882 VAL_145 2.079151 LEU_146 13.703363 VAL_147
2.690253 VAL_148 1.074774 ALA_149 0.000000 ALA_150 4.356600 SER_151
0.000000 GLY_152 12.628590 ASN_153 84.248703 SER_154 77.662354
GLY_155 25.409861 ALA_156 38.074570 GLY_157 40.493744 SER_158
53.915291 ILE_159 4.352278 SER_160 12.458543 TYR_161 29.670284
PRO_162 4.030401 ALA_163 0.968253 ARG_164 84.059120 TYR_165
28.641129 ALA_166 68.193314 ASN_167 61.686481 ALA_168 0.537387
MET_169 0.586837 ALA_170 0.000000 VAL_171 0.000000 GLY_172 0.000000
ALA_173 0.933982 THR_174 3.013133 ASP_175 34.551376 GLN_176
96.873039 ASN_177 98.664368 ASN_178 41.197159 ASN_179 60.263512
ARG_180 64.416336 ALA_181 7.254722 SER_182 91.590881 PHE_183
52.126518 SER_184 2.101459 GLN_185 15.736279 TYR_186 44.287792
GLY_187 5.114592 ALA_188 69.406563 GLY_189 36.926083 LEU_190
16.511177 ASP_191 7.705349 ILE_192 0.268693 VAL_193 4.299094
ALA_194 0.000000 PRO_195 0.806080 GLY_196 0.000000 VAL_197
25.257177 ASN_198 82.177422 VAL_199 10.747736 GLN_200 80.374527
SER_201 2.008755 THR_202 0.000000 TYR_203 80.679886 PRO_204
34.632195 GLY_205 74.536827 SER_206 74.964920 THR_207 57.070065
TYR_208 82.895500 ALA_209 22.838940 SER_210 69.045639 LEU_211
49.708279 ASN_212 86.905457 GLY_213 2.686934 THR_214 4.669909
SER_215 15.225292 MET_216 7.261287 ALA_217 0.000000 THR_218
0.000000 PRO_219 0.806080 HIS_220 2.662697 VAL_221 0.268693 ALA_222
0.000000 GLY_223 0.000000 ALA_224 7.206634 ALA_225 1.039576 ALA_226
0.268693 LEU_227 1.074774 VAL_228 1.541764 LYS_229 39.262505
GLN_230 54.501614 LYS_231 81.154129 ASN_232 30.004124 PRO_233
91.917931 SER_234 102.856705 TRP_235 64.639481 SER_236 51.797619
ASN_237 24.866917 VAL_238 78.458466 GLN_239 73.981461 ILE_240
14.474245 ARG_241 41.242931 ASN_242 64.644814 HIS_243 50.671440
LEU_244 5.127482 LYS_245 48.820000 ASN_246 115.264534 THR_247
22.205376 ALA_248 16.415077 THR_249 60.503101 SER_250 74.511597
LEU_251 48.861599 GLY_252 39.124340 SER_253 49.811481 THR_254
88.421982 ASN_255 72.490181 LEU_256 54.835758 TYR_257 38.798912
GLY_258 3.620916 SER_259 35.017368 GLY_260 0.537387 LEU_261
8.598188 VAL_262 4.519700 ASN_263 16.763659 ALA_264 3.413124
GLU_265 37.942276 ALA_266 15.871746 ALA_267 3.947115 THR_268
2.475746 ARG_269 176.743362 ION_270 0.000000 ION_271 5.197493
Subset REST: restmole.list Subset REST: SAVI8: E5-E15, E17-E18,
E22, E38-E40, E42-E43, E73-E76, E82-E86, E103-E105, SAVI8:
E108-E109, E111-E112, E115-E116, E122, E128-E144, E149-E150,
E156-E157, SAVI8: E160-E162, E165-E168, E170-E171, E173, E180-E188,
E190-E192, E200, SAVI8: E203-E204, E206, E211-E213, E215-E216,
E227-E230, E255-E259, E261-E262, SAVI8: E267-E269 restatom.list
Subset REST: SAVI8: PRO E5: N, CD, CA, CG, CB, C, O SAVI8: TRP E6:
N, CA, CD2, CE2, NE1, CD1, CG, CE3, CZ3, CH2, CZ2, CB, C, O SAVI8:
GLY E7: N, CA, C, O SAVI8: ILE E8: N, CA, CD1, CG1, CB, CG2, C, O
SAVI8: SER E9: N, CA, OG, CB, C, O SAVI8: ARG E10: N, CA, NH2, NH1,
CZ, NE, CD, CG, CB, C, O SAVI8: VAL E11: N, CA, CG2, CG1, CB, C, O
SAVI8: GLN E12: N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: ALA E13:
N, CA, CB, C, O SAVI8: PRO E14: N, CD, CA, CG, CB, C, O SAVI8: ALA
E15: N, CA, CB, C, O SAVI8: HIS E17: N, CA, CD2, NE2, CE1, ND1, CG,
CB, C, O SAVI8: ASN E18: N, CA, ND2, OD1, CG, CB, C, O SAVI8: THR
E22: N, CA, CG2, OG1, CB, C, O SAVI8: THR E38: N, CA, CG2, OG1, CB,
C, O SAVI8: HIS E39: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O SAVI8:
PRO E40: N, CD, CA, CG, CB, C, O SAVI8: LEU E42: N, CA, CD2, CD1,
CG, CB, C, O SAVI8: ASN E43: N, CA, ND2, OD1, CG, CB, C, O SAVI8:
ALA E73: N, CA, CB, C, O SAVI8: ALA E74: N, CA, CB, C, O SAVI8: LEU
E75: N, CA, CD2, CD1, CG, CB, C, O SAVI8: ASN E76: N, CA, ND2, OD1,
CG, CB, C, O SAVI8: LEU E82: N, CA, CD2, CD1, CG, CB, C, O SAVI8:
GLY E83: N, CA, C, O SAVI8: VAL E84: N, CA, CG2, CG1, CB, C, O
SAVI8: ALA E85: N, CA, CB, C, O SAVI8: PRO E86: N, CD, CA, CG, CB,
C, O SAVI8: SER E103: N, CA, OG, CB, C, O SAVI8: VAL E104: N, CA,
CG2, CG1, CB, C, O SAVI8: SER E105: N, CA, OG, CB, C, O SAVI8: ALA
E108: N, CA, CB, C, O SAVI8: GLN E109: N, CA, NE2, OE1, CD, CG, CB,
C, O SAVI8: LEU E111: N, CA, CD2, CD1, CG, CB, C, O SAVI8: GLU
E112: N, CA, OE2, OE1, CD, CG, CB, C, O SAVI8: GLY E115: N, CA, C,
O SAVI8: ASN E116: N, CA, ND2, OD1, CG, CB, C, O SAVI8: ALA E122:
N, CA, CB, C, O SAVI8: SER E128: N, CA, OG, CB, C, O SAVI8: PRO
E129: N, CD, CA, CG, CB, C, O SAVI8: SER E130: N, CA, OG, CB, C, O
SAVI8: PRO E131: N, CD, CA, CG, CB, C, O SAVI8: SER E132: N, CA,
OG, CB, C, O SAVI8: ALA E133: N, CA, CB, C, O SAVI8: THR E134: N,
CA, CG2, OG1, CB, C, O SAVI8: LEU E135: N, CA, CD2, CD1, CG, CB, C,
O SAVI8: GLU E136: N, CA, OE2, OE1, CD, CG, CB, C, O SAVI8: GLN
E137: N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: ALA E138: N, CA, CB,
C, O SAVI8: VAL E139: N, CA, CG2, CG1, CB, C, O SAVI8: ASN E140: N,
CA, ND2, OD1, CG, CB, C, O SAVI8: SER E141: N, CA, OG, CB, C, O
SAVI8: ALA E142: N, CA, CB, C, O SAVI8: THR E143: N, CA, CG2, OG1,
CB, C, O SAVI8: SER E144: N, CA, OG, CB, C, O SAVI8: VAL E149: N,
CA, CG2, CG1, CB, C, O SAVI8: VAL E150: N, CA, CG2, CG1, CB, C, O
SAVI8: SER E156: N, CA, OG, CB, C, O SAVI8: GLY E157: N, CA, C, O
SAVI8: ALA E160: N, CA, CB, C, O SAVI8: GLY E161: N, CA, C, O
SAVI8: SER E162: N, CA, OG, CB, C, O SAVI8: ILE E165: N, CA, CD1,
CG1, CB, CG2, C, O SAVI8: SER E166: N, CA, OG, CB, C, O SAVI8: TYR
E167: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: PRO
E168: N, CD, CA, CG, CB, C, O SAVI8: ARG E170: N, CA, NH2, NH1, CZ,
NE, CD, CG, CB, C, O SAVI8: TYR E171: N, CA, OH, CZ, CD2, CE2, CE1,
CD1, CG, CB, C, O SAVI8: ASN E173: N, CA, ND2, OD1, CG, CB, C, O
SAVI8: THR E180: N, CA, CG2, OG1, CB, C, O SAVI8: ASP E181: N, CA,
OD2, OD1, CG, CB, C, O SAVI8: GLN E182: N, CA, NE2, OE1, CD, CG,
CB, C, O SAVI8: ASN E183: N, CA, ND2, OD1, CG, CB, C, O SAVI8: ASN
E184: N, CA, ND2, OD1, CG, CB, C, O SAVI8: ASN E185: N, CA, ND2,
OD1, CG, CB, C, O SAVI8: ARG E186: N, CA, NH2, NH1, CZ, NE, CD, CG,
CB, C, O SAVI8: ALA E187: N, CA, CB, C, O SAVI8: SER E188: N, CA,
OG, CB, C, O SAVI8: SER E190: N, CA, OG, CB, C, O SAVI8: GLN E191:
N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: TYR E192: N, CA, OH, CZ,
CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: ALA E200: N, CA, CB, C, O
SAVI8: VAL E203: N, CA, CG2, CG1, CB, C, O SAVI8: ASN E204: N, CA,
ND2, OD1, CG, CB, C, O SAVI8: GLN E206: N, CA, NE2, OE1, CD, CG,
CB, C, O SAVI8: GLY E211: N, CA, C, O SAVI8: SER E212: N, CA, OG,
CB, C, O SAVI8: THR E213: N, CA, CG2, OG1, CB, C, O SAVI8: ALA
E215: N, CA, CB, C, O SAVI8: SER E216: N, CA, OG, CB, C, O SAVI8:
VAL E227: N, CA, CG2, CG1, CB, C, O SAVI8: ALA E228: N, CA, CB, C,
O SAVI8: GLY E229: N, CA, C, O SAVI8: ALA E230: N, CA, CB, C, O
SAVI8: THR E255: N, CA, CG2, OG1, CB, C, O SAVI8: SER E256: N, CA,
OG, CB, C, O SAVI8: LEU E257: N, CA, CD2, CD1, CG, CB, C, O SAVI8:
GLY E258: N, CA, C, O SAVI8: SER E259: N, CA, OG, CB, C, O SAVI8:
ASN E261: N, CA, ND2, OD1, CG, CB, C, O SAVI8: LEU E262: N, CA,
CD2, CD1, CG, CB, C, O SAVI8: LEU E267: N, CA, CD2, CD1, CG, CB, C,
O SAVI8: VAL E268: N, CA, CG2, CG1, CB, C, O SAVI8: ASN E269: N,
CA, ND2, OD1, CG, CB, C, O Subset SUB5B: sub5bmole.list Subset
SUB5B: SAVI8: E2-E4, E16, E19-E21, E23-E24, E28, E37, E41, E44-E45,
E77-E81, E87-E88, SAVI8: E90, E113-E114, E117-E118, E120-E121,
E145-E148, E169, E172, E174-E176, SAVI8: E193-E196, E198-E199,
E214, E231-E234, E236, E243, E247, E250, E253-E254, SAVI8: E260,
E263-E266, E270-E273, M276H-M277H sub5batom.list Subset SUB5B:
SAVI8: GLN E2: N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: SER E3: N,
CA, OG, CB, C, O SAVI8: VAL E4: N, CA, CG2, CG1, CB, C, O SAVI8:
ALA E16: N, CA, CB, C, O SAVI8: ARG E19: N, CA, NH2, NH1, CZ, NE,
CD, CG, CB, C, O SAVI8: GLY E20: N, CA, C, O SAVI8: LEU E21: N, CA,
CD2, CD1, CG, CB, C, O SAVI8: GLY E23: N, CA, C, O SAVI8: SER E24:
N, CA, OG, CB, C, O SAVI8: VAL E28: N, CA, CG2, CG1, CB, C, O
SAVI8: SER E37: N, CA, OG, CB, C, O SAVI8: ASP E41: N, CA, OD2,
OD1, CG, CB, C, O SAVI8: ILE E44: N, CA, CD1, CG1, CB, CG2, C, O
SAVI8: ARG E45: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O SAVI8:
ASN E77: N, CA, ND2, OD1, CG, CB, C, O SAVI8: SER E78: N, CA, OG,
CB, C, O SAVI8: ILE E79: N, CA, CD1, CG1, CB, CG2, C, O SAVI8: GLY
E80: N, CA, C, O SAVI8: VAL E81: N, CA, CG2, CG1, CB, C, O SAVI8:
SER E87: N, CA, OG, CB, C, O SAVI8: ALA E88: N, CA, CB, C, O SAVI8:
LEU E90: N, CA, CD2, CD1, CG, CB, C, O SAVI8: TRP E113: N, CA, CD2,
CE2, NE1, CD1, CG, CE3, CZ3, CH2, CZ2, CB, C, O SAVI8: ALA E114: N,
CA, CB, C, O SAVI8: ASN E117: N, CA, ND2, OD1, CG, CB, C, O SAVI8:
GLY E118: N, CA, C, O SAVI8: HIS E120: N, CA, CD2, NE2, CE1, ND1,
CG, CB, C, O SAVI8: VAL E121: N, CA, CG2, CG1, CB, C, O SAVI8: ARG
E145: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O SAVI8: GLY E146: N,
CA, C, O SAVI8: VAL E147: N, CA, CG2, CG1, CB, C, O SAVI8: LEU
E148: N, CA, CD2, CD1, CG, CB, C, O SAVI8: ALA E169: N, CA, CB, C,
O SAVI8: ALA E172: N, CA, CB, C, O SAVI8: ALA E174: N, CA, CB, C, O
SAVI8: MET E175: N, CA, CE, SD, CG, CB, C, O SAVI8: ALA E176: N,
CA, CB, C, O SAVI8: GLY E193: N, CA, C, O SAVI8: ALA E194: N, CA,
CB, C, O SAVI8: GLY E195: N, CA, C, O SAVI8: LEU E196: N, CA, CD2,
CD1, CG, CB, C, O SAVI8: ILE E198: N, CA, CD1, CG1, CB, CG2, C, O
SAVI8: VAL E199: N, CA, CG2, CG1, CB, C, O SAVI8: TYR E214: N, CA,
OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: ALA E231: N, CA,
CB, C, O SAVI8: ALA E232: N, CA, CB, C, O SAVI8: LEU E233: N, CA,
CD2, CD1, CG, CB, C, O SAVI8: VAL E234: N, CA, CG2, CG1, CB, C, O
SAVI8: GLN E236: N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: ASN E243:
N, CA, ND2, OD1, CG, CB, C, O SAVI8: ARG E247: N, CA, NH2, NH1, CZ,
NE, CD, CG, CB, C, O SAVI8: LEU E250: N, CA, CD2, CD1, CG, CB, C, O
SAVI8: THR E253: N, CA, CG2, OG1, CB, C, O SAVI8: ALA E254: N, CA,
CB, C, O SAVI8: THR E260: N, CA, CG2, OG1, CB, C, O SAVI8: TYR
E263: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: GLY
E264: N, CA, C, O SAVI8: SER E265: N, CA, OG, CB, C, O SAVI8: GLY
E266: N, CA, C, O SAVI8: ALA E270: N, CA, CB, C, O SAVI8: GLU E271:
N, CA, OE2, OE1, CD, CG, CB, C, O SAVI8: ALA E272: N, CA, CB, C, O
SAVI8: ALA E273: N, CA, CB, C, O SAVI8: ION M276H: CA SAVI8: ION
M277H: CA Subset ACTSITE: actsitemole.list Subset ACTSITE: SAVI8:
E29-E35, E48-E51, E54, E58-E72, E91-E102, E106-E107, E110,
E123-E127, SAVI8: E151-E155, E177-E179, E189, E201-E202, E205,
E207-E210, E217-E226 actsiteatom.list Subset ACTSITE: SAVI8: ALA
E29: N, CA, CB, C, O SAVI8: VAL E30: N, CA, CG2, CG1, CB, C, O
SAVI8: LEU E31: N, CA, CD2, CD1, CG, CB, C, O SAVI8: ASP E32: N,
CA, OD2, OD1, CG, CB, C, O SAVI8: THR E33: N, CA, CG2, OG1, CB, C,
O SAVI8: GLY E34: N, CA, C, O SAVI8: ILE E35: N, CA, CD1, CG1, CB,
CG2, C, O SAVI8: ALA E48: N, CA, CB, C, O SAVI8: SER E49: N, CA,
OG, CB, C, O SAVI8: PHE E50: N, CA, CD2, CE2, CZ, CE1, CD1, CG, CB,
C, O SAVI8: VAL E51: N, CA, CG2, CG1, CB, C, O SAVI8: GLU E54: N,
CA, OE2, OE1, CD, CG, CB, C, O SAVI8: THR E58: N, CA, CG2, OG1, CB,
C, O SAVI8: GLN E59: N, CA, NE2, OE1, CD, CG, CB, C, O SAVI8: ASP
E60: N, CA, OD2, OD1, CG, CB, C, O SAVI8: GLY E61: N, CA, C, O
SAVI8: ASN E62: N, CA, ND2, OD1, CG, CB, C, O SAVI8: GLY E63: N,
CA, C, O SAVI8: HIS E64: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
SAVI8: GLY E65: N, CA, C, O SAVI8: THR E66: N, CA, CG2, OG1, CB, C,
O SAVI8: HIS E67: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O SAVI8:
VAL E68: N, CA, CG2, CG1, CB, C, O SAVI8: ALA E69: N, CA, CB, C, O
SAVI8: GLY E70: N, CA, C, O SAVI8: THR E71: N, CA, CG2, OG1, CB, C,
O SAVI8: ILE E72: N, CA, CD1, CG1, CB, CG2, C, O SAVI8: TYR E91: N,
CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: ALA E92: N, CA,
CB, C, O SAVI8: VAL E93: N, CA, CG2, CG1, CB, C, O SAVI8: LYS E94:
N, CA, NZ, CE, CD, CG, CB, C, O SAVI8: VAL E95: N, CA, CG2, CG1,
CB, C, O SAVI8: LEU E96: N, CA, CD2, CD1, CG, CB, C, O SAVI8: GLY
E97: N, CA, C, O SAVI8: ALA E98: N, CA, CB, C, O SAVI8: SER E99: N,
CA, OG, CB, C, O SAVI8: GLY E100: N, CA, C, O SAVI8: SER E101: N,
CA, OG, CB, C, O SAVI8: GLY E102: N, CA, C, O SAVI8: SER E106: N,
CA, OG, CB, C, O SAVI8: ILE E107: N, CA, CD1, CG1, CB, CG2, C, O
SAVI8: GLY E110: N, CA, C, O SAVI8: ASN E123: N, CA, ND2, OD1,
CG,
CB, C, O SAVI8: LEU E124: N, CA, CD2, CD1, CG, CB, C, O SAVI8: SER
E125: N, CA, OG, CB, C, O SAVI8: LEU E126: N, CA, CD2, CD1, CG, CB,
C, O SAVI8: GLY E127: N, CA, C, O SAVI8: ALA E151: N, CA, CB, C, O
SAVI8: ALA E152: N, CA, CB, C, O SAVI8: SER E153: N, CA, OG, CB, C,
O SAVI8: GLY E154: N, CA, C, O SAVI8: ASN E155: N, CA, ND2, OD1,
CG, CB, C, O SAVI8: VAL E177: N, CA, CG2, CG1, CB, C, O SAVI8: GLY
E178: N, CA, C, O SAVI8: ALA E179: N, CA, CB, C, O SAVI8: PHE E189:
N, CA, CD2, CE2, CZ, CE1, CD1, CG, CB, C, O SAVI8: PRO E201: N, CD,
CA, CG, CB, C, O SAVI8: GLY E202: N, CA, C, O SAVI8: VAL E205: N,
CA, CG2, CG1, CB, C, O SAVI8: SER E207: N, CA, OG, CB, C, O SAVI8:
THR E208: N, CA, CG2, OG1, CB, C, O SAVI8: TYR E209: N, CA, OH, CZ,
CD2, CE2, CE1, CD1, CG, CB, C, O SAVI8: PRO E210: N, CD, CA, CG,
CB, C, O SAVI8: LEU E217: N, CA, CD2, CD1, CG, CB, C, O SAVI8: ASN
E218: N, CA, ND2, OD1, CG, CB, C, O SAVI8: GLY E219: N, CA, C, O
SAVI8: THR E220: N, CA, CG2, OG1, CB, C, O SAVI8: SER E221: N, CA,
OG, CB, C, O SAVI8: MET E222: N, CA, CE, SD, CG, CB, C, O SAVI8:
ALA E223: N, CA, CB, C, O SAVI8: THR E224: N, CA, CG2, OG1, CB, C,
O SAVI8: PRO E225: N, CD, CA, CG, CB, C, O SAVI8: HIS E226: N, CA,
CD2, NE2, CE1, ND1, CG, CB, C, O Subset RESTx: restxmole.list
Subset RESTX: NEWMODEL: E5, E13-E14, E22, E38-E40, E42, E73-E76,
E82-E86, E103-E105, NEWMODEL: E108, E122, E133-E135, E137-E140,
E149-E150, E173, E204, E206, NEWMODEL: E211-E213, E215-E216,
E227-E229, E258, E269 restxatom.list Subset RESTX: NEWMODEL: PRO
E5: N, CD, CA, CG, CB, C, O NEWMODEL: ALA E13: N, CA, CB, C, O
NEWMODEL: PRO E14: N, CD, CA, CG, CB, C, O NEWMODEL: THR E22: N,
CA, CG2, OG1, CB, C, O NEWMODEL: THR E38: N, CA, CG2, OG1, CB, C, O
NEWMODEL: HIS E39: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
NEWMODEL: PRO E40: N, CD, CA, CG, CB, C, O NEWMODEL: LEU E42: N,
CA, CD2, CD1, CG, CB, C, O NEWMODEL: ALA E73: N, CA, CB, C, O
NEWMODEL: ALA E74: N, CA, CB, C, O NEWMODEL: LEU E75: N, CA, CD2,
CD1, CG, CB, C, O NEWMODEL: ASN E76: N, CA, ND2, CD1, CG, CB, C, O
NEWMODEL: LEU E82: N, CA, CD2, CD1, CG, CB, C, O NEWMODEL: GLY E83:
N, CA, C, O NEWMODEL: VAL E84: N, CA, CG2, CG1, CB, C, O NEWMODEL:
ALA E85: N, CA, CB, C, O NEWMODEL: PRO E86: N, CD, CA, CG, CB, C, O
NEWMODEL: SER E103: N, CA, OG, CB, C, O NEWMODEL: VAL E104: N, CA,
CG2, CG1, CB, C, O NEWMODEL: SER E105: N, CA, OG, CB, C, O
NEWMODEL: ALA E108: N, CA, CB, C, O NEWMODEL: ALA E122: N, CA, CB,
C, O NEWMODEL: ALA E133: N, CA, CB, C, O NEWMODEL: THR E134: N, CA,
CG2, OG1, CB, C, O NEWMODEL: LEU E135: N, CA, CD2, CD1, CG, CB, C,
O NEWMODEL: GLN E137: N, CA, NE2, OE1, CD, CG, CB, C, O NEWMODEL:
ALA E138: N, CA, CB, C, O NEWMODEL: VAL E139: N, CA, CG2, CG1, CB,
C, O NEWMODEL: ASN E140: N, CA, ND2, OD1, CG, CB, C, O NEWMODEL:
VAL E149: N, CA, CG2, CG1, CB, C, O NEWMODEL: VAL E150: N, CA, CG2,
CG1, CB, C, O NEWMODEL: ASN E173: N, CA, ND2, OD1, CG, CB, C, O
NEWMODEL: ASN E204: N, CA, ND2, OD1, CG, CB, C, O NEWMODEL: GLN
E206: N, CA, NE2, OE1, CD, CG, CB, C, O NEWMODEL: GLY E211: N, CA,
C, O NEWMODEL: SER E212: N, CA, OG, CB, C, O NEWMODEL: THR E213: N,
CA, CG2, OG1, CB, C, O NEWMODEL: ALA E215: N, CA, CB, C, O
NEWMODEL: SER E216: N, CA, OG, CB, C, O NEWMODEL: VAL E227: N, CA,
CG2, CG1, CB, C, O NEWMODEL: ALA E228: N, CA, CB, C, O NEWMODEL:
GLY E229: N, CA, C, O NEWMODEL: GLY E258: N, CA, C, O NEWMODEL: ASN
E269: N, CA, ND2, OD1, CG, CB, C, O
Example 3
[0548] Suitable Substitutions in PD498 for Addition of Carboxylic
Acid Attachment Groups (--COOH)
[0549] The 3D structure of PD498 was modeled as described in
Example 1. Suitable locations for addition of carboxylic attachment
groups (aspartatic acids and glutamic acids) were found as follows.
The procedure described in Example 1 was followed. The commands
performed in Insight (BIOSYM) are shown in the command files
makeDEzone.bcl and makeDEzone2.bcl below:
[0550] Conservative Substutitions:
[0551] makeDEzone.bcl
[0552] Delete Subset *
[0553] Color Molecule Atoms * Specified Specification 255,0,255
[0554] Zone Subset ASP :asp:od* Static monomer/residue 10
Color_Subset 255,255,0
[0555] Zone Subset GLU :glu:oe* Static monomer/residue 10
Color_Subset 255,255,0
[0556] #NOTE: editnextline C-terminal residue number according to
the protein
[0557] Zone Subset CTERM :280:O Static monomer/residue 10
Color_Subset 255,255,0
[0558] #NOTE: editnextline ACTSITE residues according to the
protein
[0559] Zone Subset ACTSITE :39,72,226 Static monomer/residue 8
Color_Subset 255,255,0
[0560] Combine Subset ALLZONE Union ASP GLU
[0561] Combine Subset ALLZONE Union ALLZONE CTERM
[0562] Combine Subset ALLZONE Union ALLZONE ACTSITE
[0563] #NOTE: editnextline object name according to the protein
[0564] Combine Subset REST Difference PD498FINALMODEL ALLZONE
[0565] List Subset REST Atom Output_File restatom.list
[0566] List Subset REST monomer/residue Output_File
restmole.list
[0567] Color Molecule Atoms ACTSITE Specified Specification
255,0,0
[0568] List Subset ACTSITE Atom Output_File actsiteatom.list
[0569] List Subset ACTSITE monomer/residue Output_File
actsitemole.list
[0570] #
[0571] Zone Subset REST5A REST Static Monomer/Residue
5-Color_Subset
[0572] Combine Subset SUB5A Difference REST5A ACTSITE
[0573] Combine Subset SUB5B Difference SUB5A REST
[0574] Color Molecule Atoms SUB5B Specified Specification
255,255,255
[0575] List Subset SUB5B Atom Output_File sub5batom.list
[0576] List Subset SUB5B monomer/residue Output_File
sub5bmole.list
[0577] #Now identify sites for asn->asp & gln->glu
substitutions and . . .
[0578] #continue with makezone2.bcl.
[0579] #Use grep command to identify asn/gln in restatom.list . .
.
[0580] #sub5batom.list & accsiteatom.list.
[0581] Comments:
[0582] The subset REST contains Gln33 and Asn245, SUB5B contains
Gln12, Gln126, Asn209, Gln242, Asn246, Gln248 and Asn266, all of
which are solvent exposed.
[0583] The substitutions Q12E or Q12D, Q33E or Q33D, Q126E or
Q126D, N209D or N209E, Q242E or Q242D, N245D or N245E, N246D or
N246E, Q248E or Q248D and N266D or N266E are identified in PD498 as
sites for mutagenesis within the scope of this invention. Residues
are substituted below in section 2, and further analysis done:
[0584] Non-Conservative Substitutions:
[0585] makeDEzone2.bcl
[0586] #sourcefile makezone2.bcl Claus von der Osten 961128
[0587] #
[0588] #having scanned lists (grep gln/asn command) and identified
sites for
[0589] #asn->asp & gln->glu substitutions
[0590] #NOTE: editnextline object name according to protein
[0591] Copy Object -To_Clipboard -Displace PD498FINALMODEL
newmodel
[0592] Biopolymer
[0593] #NOTE: editnextline object name according to protein
[0594] Blank Object On PD498FINALMODEL
[0595] #NOTE: editnextlines with asn->asp & gln->glu
positions
[0596] Replace Residue newmodel:33 glu L
[0597] Replace Residue newmodel:245 asp L
[0598] Replace Residue newmodel:12 glu L
[0599] Replace Residue newmodel:126 glu L
[0600] Replace Residue newmodel:209 asp L
[0601] Replace Residue newmodel:242 glu L
[0602] Replace Residue newmodel:246 asp L
[0603] Replace Residue newmodel:248 glu L
[0604] Replace Residue newmodel:266 asp L
[0605] #
[0606] #Now repeat analysis done prior to asn->asp &
gln->glu,
[0607] #now including introduced asp & glu
[0608] Color Molecule Atoms newmodel Specified Specification
255,0,255
[0609] Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10
Color_Subset 255,255,0
[0610] Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10
Color_Subset 255,255,0
[0611] #NOTE: editnextline C-terminal residue number according to
the protein
[0612] Zone Subset CTERMx newmodel:280:O Static monomer/residue
10
[0613] Color_Subset 255,255,0
[0614] #NOTE: editnextline ACTSITEx residues according to the
protein
[0615] Zone Subset ACTSITEx newmodel:39,72,226 Static
monomer/residue 8 Color_Subset 255,255,0
[0616] Combine Subset ALLZONEx Union ASPx GLUx
[0617] Combine Subset ALLZONEx Union ALLZONEx CTERMx
[0618] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
[0619] Combine Subset RESTx Difference newmodel ALLZONEx
[0620] List Subset RESTx Atom Output_File restxatom.list
[0621] List Subset RESTx monomer/residue Output_File
restxmole.list
[0622] #
[0623] Color Molecule Atoms ACTSITEx Specified Specification
255,0,0
[0624] List Subset ACTSITEx Atom Output_File actsitexatom.list
[0625] List Subset ACTSITEx monomer/residue Output_File
actsitexmole.list
[0626] #
[0627] #read restxatom.list or restxmole.list to identify sites for
(not_gluasp)->gluasp . . .
[0628] #subst. if needed.
[0629] Comments:
[0630] The subset RESTx contains only two residues: A233 and G234,
none of which are solvent exposed. No further mutagenesis is
required to obtain complete protection of the surface. However, it
may be necessary to remove some of the reactive carboxylic groups
in the active site region to ensure access to the active site of
PD498. Acidic residues within the subset ACTSITE are: D39, D58, D68
and D106. Of these only the two latter are solvent exposed and D39
is a functional residue. The mutations D68N, D68Q, D106N and D106Q
were found suitable according to the present invention.
[0631] Relevant Data for Example 3:
[0632] Solvent Accessibility Data for PD498MODEL: see Example 1
Above.
9 Subset REST: restmole.list Subset REST: PD498FINALMODEL: 10-11,
33-35, 54-55, 129-130, 221, 233-234, 236, 240, 243,
PD498FINALMODEL: 245, 262, 264-265 restatom.list Subset REST:
PD498FINALMODEL: ALA 10: N, CA, C, O, CB PD498FINALMODEL: TYR 11:
N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH PD498FINALMODEL:
GLN 33: N, CA, C, O, CB, CG, CD, OE1, NE2 PD498FINALMODEL: THR 34:
N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL: VAL 35: N, CA, C, O, CB,
CG1, CG2 PD498FINALMODEL: ILE 54: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: LYS 55: N, CA, C, O, CB, CG, CD, CE, NZ
PD498FINALMODEL: LYS 129: N, CA, C, O, CB, CG, CD, CE, NZ
PD498FINALMODEL: VAL 130: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: ALA 233: N, CA, C, O, CB PD498FINALMODEL:
GLY 234: N, CA, C, O PD498FINALMODEL: ALA 236: N, CA, C, O, CB
PD498FINALMODEL: ALA 240: N, CA, C, O, CB PD498FINALMODEL: GLY 243:
N, CA, C, O PD498FINALMODEL: ASN 245: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: GLY 262: N, CA, C, O PD498FINALMODEL: GLY 264: N,
CA, C, O PD498FINALMODEL: THR 265: N, CA, C, O, CB, OG1, CG2 Subset
SUB5B: sub5bmole.list Subset SUB5B: PD498FINALMODEL: 6-9, 12-13,
31-32, 51-53, 56, 81, 93-94, 97-99, 122, 126-128, PD498FINALMODEL:
131, 155-157, 159, 197-199, 209, 211, 219-220, 232, 235,
PD498FINALMODEL: 237-239, 241-242, 244, 246-249, 253, 260-261, 263,
266-268 sub5batom.list Subset SUB5B: PD498FINALMODEL: PRO 6: N, CA,
CD, C, O, CB, CG PD498FINALMODEL: TYR 7: N, CA, C, O, CB, CG, CD1,
CD2, CE1, CE2, CZ, OH PD498FINALMODEL: TYR 8: N, CA, C, O, CB, CG,
CD1, CD2, CE1, CE2, CZ, OH PD498FINALMODEL: SER 9: N, CA, C, O, CB,
OG PD498FINALMODEL: GLN 12: N, CA, C, O, CB, CG, CD, OE1, NE2
PD498FINALMODEL: TYR 13: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: SER 31: N, CA, C, O, CB, OG
PD498FINALMODEL: THR 32: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL:
ARG 51: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 PD498FINALMODEL:
LYS 52: N, CA, C, O, CB, CG, CD, CE, NZ PD498FINALMODEL: VAL 53: N,
CA, C, O, CB, CG1, CG2 PD498FINALMODEL: GLY 56: N, CA, C, O
PD498FINALMODEL: ALA 81: N, CA, C, O, CB PD498FINALMODEL: MET 93:
N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: ALA 94: N, CA, C, O,
CB PD498FINALMODEL: THR 97: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: LYS 98: N, CA, C, O, CB, CG, CD, CE, NZ
PD498FINALMODEL: ILE 99: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: TYR 122: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: GLN 126: N, CA, C, O, CB, CG, CD, OE1, NE2
PD498FINALMODEL: GLY 127: N, CA, C, O PD498FINALMODEL: ALA 128: N,
CA, C, O, CB PD498FINALMODEL: LEU 131: N, CA, C, O, CB, CG, CD1,
CD2 PD498FINALMODEL: GLY 155: N, CA, C, O PD498FINALMODEL: ALA 156:
N, CA, C, O, CB PD498FINALMODEL: VAL 157: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: VAL 159: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: TYR 197: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: GLY 198: N, CA, C, O PD498FINALMODEL: THR
199: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL: ASN 209: N, CA, C,
O, CB, CG, OD1, ND2 PD498FINALMODEL: ALA 211: N, CA, C, O, CB
PD498FINALMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: SER 220: N, CA, C, O, CB, OG
PD498FINALMODEL: VAL 232: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: LEU 235: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ALA 237: N, CA, C, O, CB PD498FINALMODEL: LEU 238:
N, CA, C, O, CB, CG, CD1, CD2 PD498FINALMODEL: LEU 239: N, CA, C,
O, CB, CG, CD1, CD2 PD498FINALMODEL: SER 241: N, CA, C, O, CB, OG
PD498FINALMODEL: GLN 242: N, CA, C, O, CB, CG, CD, OE1, NE2
PD498FINALMODEL: LYS 244: N, CA, C, O, CB, CG, CD, CE, NZ
PD498FINALMODEL: ASN 246: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: VAL 247: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: GLN 248: N, CA, C, O, CB, CG, CD, OE1, NE2
PD498FINALMODEL: ILE 249: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: ILE 253: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: ILE 260: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: SER 261: N, CA, C, O, CB, OG PD498FINALMODEL: THR
263: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL: ASN 266: N, CA, C,
O, CB, CG, OD1, ND2 PD498FINALMODEL: PHE 267: N, CA, C, O, CB, CG,
CD1, CD2, CE1, CE2, CZ PD498FINALMODEL: LYS 268: N, CA, C, O, CB,
CG, CD, CE, NZ Subset ACTSITE: actsitemole.list Subset ACTSITE:
PD498FINALMODEL: 36-42, 57-60, 66-80, 100-110, 115-116, 119,
132-136, 160-164, PD498FINALMODEL: 182-184, 194, 206-207, 210,
212-215, 222-231 actsiteatom.list Subset ACTSITE: PD498FINALMODEL:
ALA 36: N, CA, C, O, CB PD498FINALMODEL: VAL 37: N, CA, C, O, CB,
CG1, CG2 PD498FINALMODEL: LEU 38: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ASP 39: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: SER 40: N, CA, C, O, CB, OG PD498FINALMODEL: GLY
41: N, CA, C, O PD498FINALMODEL: VAL 42: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: TYR 57: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ, OH PD498FINALMODEL: ASP 58: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: PHE 59: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ PD498FINALMODEL: ILE 60: N, CA, C, O, CB, CG1, CG2, CD1
PD498FINALMODEL: PRO 66: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
MET 67: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: ASP 68: N, CA,
C, O, CB, CG, OD1, OD2 PD498FINALMODEL: LEU 69: N, CA, C, O, CB,
CG, CD1, CD2 PD498FINALMODEL: ASN 70: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: GLY 71: N, CA, C, O PD498FINALMODEL: HIS 72: N,
CA, C, O, CB, CG, ND1, CD2, CE1, NE2 PD498FINALMODEL: GLY 73: N,
CA, C, O PD498FINALMODEL: THR 74: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: HIS 75: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
PD498FINALMODEL: VAL 76: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL:
ALA 77: N, CA, C, O, CB PD498FINALMODEL: GLY 78: N, CA, C, O
PD498FINALMODEL: THR 79: N, CA, C, O, CB, OG1, CG2 PD498FINALMODEL:
VAL 80: N, CA, C, O, CB, CG1, CG2 PD498FINALMODEL: LEU 100: N, CA,
C, O, CB, CG, CD1, CD2 PD498FINALMODEL: ALA 101: N, CA, C, O, CB
PD498FINALMODEL: VAL 102: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: ARG 103: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
PD498FINALMODEL: VAL 104: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: LEU 105: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: ASP 106: N, CA, C, O, CB, CG, OD1, OD2
PD498FINALMODEL: ALA 107: N, CA, C, O, CB PD498FINALMODEL: ASN 108:
N, CA, C, O, CB, CG, OD1, ND2 PD498FINALMODEL: GLY 109: N, CA, C, O
PD498FINALMODEL: SER 110: N, CA, C, O, CB, OG PD498FINALMODEL: SER
115: N, CA, C, O, CB, OG PD498FINALMODEL: ILE 116: N, CA, C, O, CB,
CG1, CG2, CD1 PD498FINALMODEL: GLY 119: N, CA, C, O
PD498FINALMODEL: ASN 132: N, CA, C, O, CB, CG, OD1, ND2
PD498FINALMODEL: LEU 133: N, CA, C, O, CB, CG, CD1, CD2
PD498FINALMODEL: SER 134: N, CA, C, O, CB, OG PD498FINALMODEL: LEU
135: N, CA, C, O, CB, CG, CD1, CD2 PD498FINALMODEL: GLY 136: N, CA,
C, O PD498FINALMODEL: ALA 160: N, CA, C, O, CB PD498FINALMODEL: ALA
161: N, CA, C, O, CB PD498FINALMODEL: ALA 162: N, CA, C, O, CB
PD498FINALMODEL: GLY 163: N, CA, C, O PD498FINALMODEL: ASN 164: N,
CA, C, O, CB, CG, OD1, ND2 PD498FINALMODEL: VAL 182: N, CA, C, O,
CB, CG1, CG2 PD498FINALMODEL: GLY 183: N, CA, C, O PD498FINALMODEL:
ALA 184: N, CA, C, O, CB PD498FINALMODEL: PHE 194: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ PD498FINALMODEL: PRO 206: N, CA, CD, C,
O, CB, CG PD498FINALMODEL: GLY 207: N, CA, C, O PD498FINALMODEL:
ILE 210: N, CA, C, O, CB, CG1, CG2, CD1 PD498FINALMODEL: SER 212:
N, CA, C, O, CB, OG PD498FINALMODEL: THR 213: N, CA, C, O, CB, OG1,
CG2 PD498FINALMODEL: VAL 214: N, CA, C, O, CB, CG1, CG2
PD498FINALMODEL: PRO 215: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
MET 222: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: SER 223: N,
CA, C, O, CB, OG PD498FINALMODEL: GLY 224: N, CA, C, O
PD498FINALMODEL: THR 225: N, CA, C, O, CB, OG1, CG2
PD498FINALMODEL: SER 226: N, CA, C, O, CB, OG PD498FINALMODEL: MET
227: N, CA, C, O, CB, CG, SD, CE PD498FINALMODEL: ALA 228: N, CA,
C, O, CB PD498FINALMODEL: SER 229: N, CA, C, O, CB, OG
PD498FINALMODEL: PRO 230: N, CA, CD, C, O, CB, CG PD498FINALMODEL:
HIS 231: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2 Subset RESTx:
restxmole.list Subset RESTX: NEWMODEL: 233-234 restxatom.list
Subset RESTX: NEWMODEL: ALA 233: N, CA, C, O, CB NEWMODEL: GLY 234:
N, CA, C, O
Example 4
[0633] Suitable substitutions in the Arthromyces ramosus peroxidase
for addition of carboxylic acid attachment groups (--COOH) Suitable
locations for addition of carboxylic attachment groups (aspartatic
acids and glutamic acids) in a non-hydrolytic enzyme, Arthromyces
ramosus peroxidase were found as follows.
[0634] The 3D structure of this oxido-reductase is available in the
Brookhaven Databank as larp.pdb. This A. ramosus peroxidase
contains 344 amino acid residues. The first eight residues are not
visible in the X-ray structure: QGPGGGGG, and N143 is
glycosylated.
[0635] The procedure described in Example 1 was followed.
[0636] The amino acid sequence of Arthromyces ramosus Peroxidase
(E.C.1.11.1.7) is shown in SEQ ID NO: 4.
[0637] The commands performed in Insight (BIOSYM) are shown in the
command files makeDEzone.bcl and makeDEzone2.bcl below. The
C-terminal residue is P344, the ACTSITE is defined as the heme
group and the two histidines coordinating it (H56 & H184).
[0638] Conservative Substitutions:
[0639] makeDEzone.bcl
[0640] Delete Subset *
[0641] Color Molecule Atoms * Specified Specification 255,0,255
[0642] Zone Subset ASP :asp:od* Static monomer/residue 10
Color_Subset 255,255,0
[0643] Zone Subset GLU :glu:oe* Static monomer/residue 10
Color_Subset 255,255,0
[0644] #NOTE: editnextline C-terminal residue number according to
the protein
[0645] Zone Subset CTERM :344:O Static monomer/residue 10
Color_Subset 255,255,0
[0646] #NOTE: editnextline ACTSITE residues according to the
protein
[0647] Zone Subset ACTSITE :HEM,56,184 Static monomer/residue 8
Color_Subset 255,255,0
[0648] Combine Subset ALLZONE Union ASP GLU
[0649] Combine Subset ALLZONE Union ALLZONE CTERM
[0650] Combine Subset ALLZONE Union ALLZONE ACTSITE
[0651] #NOTE: editnextline object name according to the protein
[0652] Combine Subset REST Difference ARP ALLZONE
[0653] List Subset REST Atom Output_File restatom.list
[0654] List Subset REST monomer/residue Output_File
restmole.list
[0655] Color Molecule Atoms ACTSITE Specified Specification
255,0,0
[0656] List Subset ACTSITE Atom Output_File actsiteatom.list
[0657] List Subset ACTSITE monomer/residue Output_File
actsitemole.list
[0658] #
[0659] Zone Subset REST5A REST Static Monomer/Residue
5-Color_Subset
[0660] Combine Subset SUB5A Difference REST5A ACTSITE
[0661] Combine Subset SUB5B Difference SUB5A REST
[0662] Color Molecule Atoms SUB5B Specified Specification
255,255,255
[0663] List Subset SUB5B Atom Output_File sub5batom.list
[0664] List Subset SUB5B monomer/residue Output_File
sub5bmole.list
[0665] #Now identify sites for asn->asp & gln->glu
substitutions and . . .
[0666] #continue with makezone2.bcl.
[0667] #Use grep command to identify asn/gln in restatom.list . .
.
[0668] #sub5batom.list & accsiteatom.list.
[0669] Comments:
[0670] The subset REST contains Gln70, and SUB5B contains Gln34,
Asn128, Asn303 all of which are solvent exposed. The substitutions
Q34E or Q34D, Q70E or Q70D, N128D or N128E and N303D or N303E are
identified in A. ramosus peroxidase as sites for mutagenesis.
Residues are substituted below and further analysis done:
[0671] Non-Conservative Substitutions:
[0672] makeDEzone2.bcl
[0673] #sourcefile makezone2.bcl Claus von der Osten 961128
[0674] #
[0675] #having scanned lists (grep gln/asn command) and identified
sites for . . .
[0676] #asn->asp & gln->glu substitutions
[0677] #NOTE: editnextline object name according to protein
[0678] Copy Object -To_Clipboard -Displace ARP newmodel
[0679] Biopolymer
[0680] #NOTE: editnextline object name according to protein
[0681] Blank Object On ARP
[0682] #NOTE: editnextlines with asn->asp & gln->glu
positions
[0683] Replace Residue newmodel:34 glu L
[0684] Replace Residue newmodel:70 glu L
[0685] Replace Residue newmodel:128 asp L
[0686] Replace Residue newmodel:303 asp L
[0687] #
[0688] #Now repeat analysis done prior to asn->asp &
gln->glu, . . .
[0689] #now including introduced asp & glu
[0690] Color Molecule Atoms newmodel Specified Specification
255,0,255
[0691] Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10
Color_Subset 255,255,0
[0692] xZone Subset GLUx newmodel:glu:oe* Static monomer/residue 10
Color_Subset 255,255,0
[0693] #NOTE: editnextline C-terminal residue number according to
the protein
[0694] Zone Subset CTERMx newmodel:344:O Static monomer/residue 10
Color_Subset 255,255,0
[0695] #NOTE: editnextline ACTSITEx residues according to the
protein
[0696] Zone Subset ACTSITEx newmodel:HEM,56,184 Static
monomer/residue 8 Color_Subset 255,255,0
[0697] Combine Subset ALLZONEx Union ASPx GLUx
[0698] Combine Subset ALLZONEx Union ALLZONEx CTERMx
[0699] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
[0700] Combine Subset RESTx Difference newmodel ALLZONEx
[0701] List Subset RESTx Atom Output_File restxatom.list
[0702] List Subset RESTx monomer/residue Output_File
restxmole.list
[0703] #
[0704] Color Molecule Atoms ACTSITEx Specified Specification
255,0,0
[0705] List Subset ACTSITEx Atom Output_File actsitexatom.list
[0706] List Subset ACTSITEx monomer/residue Output_File
actsitexmole.list
[0707] #
[0708] #read restxatom.list or restxmole.list to identify sites for
(not_gluasp)->gluasp . . .
[0709] #subst. if needed.
[0710] Comments:
[0711] The subset RESTx contains only four residues: S9, S334, G335
and P336, all of which are >5% solvent exposed. The mutations
S9D, S9E, S334D, S334E, G335D, G335E, P336D and P336E are proposed
in A. ramosus peroxidase. Acidic residues within the subset ACTSITE
are: E44, D57, D77, E87, E176, D179, E190, D202, D209, D246 and the
N-terminal carboxylic acid on P344. Of these only E44, D77, E176,
D179, E190, D209, D246 and the N-terminal carboxylic acid on P344
are solvent exposed. Suitable sites for mutations are E44Q, D77N,
E176Q, D179N, E190Q, D209N and D246N. D246N and D246E are risky
mutations due to D246's importance for binding of heme.
[0712] The N-terminal 8 residues were not included in the
calculations above, as they do not appear in the structure. None of
these 8 residues, QGPGGGG, contain carboxylic groups. The following
variants are proposed as possible mutations to enable attachment to
this region: Q1E, Q1D, G2E, G2D, P3E, P3D, G4E, G4D, G5E, G5D, G6E,
G6D, G7E, G7D, G8E, G8D.
[0713] Relevant Data for Example 4:
[0714] Solvent accessibility data for A. ramosus peroxidase (Note:
as the first eight residues are missing in the X-ray structure, the
residue numbers printed in the accessibility list below are 8 lower
than those used elsewhere for residue numbering.
10 # ARP Thu Jan 30 15:39:05 MET 1997 # residue area SER_1
143.698257 VAL_2 54.879990 THR_3 86.932701 CYS_4 8.303715 PRO_5
126.854782 GLY_6 53 .771488 GLY_7 48.137802 GLN_8 62.288475 SER_9
79.932549 THR_10 16.299215 SER_11 81.928642 ASN_12 51.432678 SER_13
81.993019 GLN_14 92.344009 CYS_15 0.000000 CYS_16 32.317432 VAL_17
54.067810 TRP_18 6.451035 PHE_19 25.852070 ASP_20 79.033997 VAL_21
0.268693 LEU_22 22.032858 ASP_23 90.111404 ASP_24 43.993240 LEU_25
1.074774 GLN_26 25.589321 THR_27 82.698059 ASN_28 96.600883 PHE_29
32.375275 TYR_30 5.898365 GLN_31 103.380585 GLY_32 40.042034 SER_33
46.789322 LYS_34 87.161873 CYS_35 12.827215 GLU_36 51.582657 SER_37
16.378180 PRO_38 33.560043 VAL_39 6.448641 ARG_40 7.068311 LYS_41
15.291286 ILE_42 1.612160 LEU_43 1.880854 ARG_44 16.906845 ILE_45
0.000000 VAL_46 2.312647 PHE_47 2.955627 HIS_48 20.392527 ASP_49
4.238116 ALA_50 0.510757 ILE_51 1.576962 GLY_52 2.858601 PHE_53
48.633503 SER_54 8.973248 PRO_55 58.822315 ALA_56 59.782852 LEU_57
46.483955 THR_58 86.744827 ALA_59 89.515816 ALA_60 81.163239 GLY_61
70.119019 GLN_62 112.635498 PHE_63 93.522354 GLY_64 2.742587 GLY_65
13.379636 GLY_66 22.722847 GLY_67 0.000000 ALA_68 0.268693 ASP_69
12.074840 GLY_70 0.700486 SER_71 0.000000 ILE_72 0.000000 ILE_73
0.000000 ALA_74 17.304443 HIS_75 41.071186 SER_76 20.000793 ASN_77
120.855316 ILE_78 66.574982 GLU_79 2.334954 LEU_80 41.329689 ALA_81
77.370575 PHE_82 38.758774 PRO_83 131.946289 ALA_84 34.893864
ASN_85 5.457000 GLY_86 43.364151 GLY_87 51.561348 LEU_88 0.242063
THR_89 73.343575 ASP_90 130.139389 THR_91 17.863211 ILE_92 0.268693
GLU_93 92.210396 ALA_94 35.445068 LEU_95 1.343467 ARG_96 31.175611
ALA_97 44.650192 VAL_98 17.698566 GLY_99 1.471369 ILE_100 62.441463
ASN_101 107.139748 HIS_102 46.952496 GLY_103 46.559296 VAL_104
11.342628 SER_105 15.225677 PHE_106 6.422011 GLY_107 3.426864
ASP_108 10.740790 LEU_109 0.268693 ILE_110 1.880854 GLN_111
31.867456 PHE_112 0.000000 ALA_113 0.000000 THR_114 3.656114
ALA_115 8.299393 VAL_116 0.268693 GLY_117 0.268693 MET_118 3.761708
SER_119 14.536770 ASN_120 25.928799 CYS_121 0.537387 PRO_122
29.798336 GLY_123 33.080013 SER_124 17.115562 PRO_125 36.908714
ARG_126 108.274727 LEU_127 21.238588 GLU_128 53.742313 PHE_129
3.761708 LEU_130 12.928699 THR_131 10.414591 GLY_132 47.266495
ARG_133 12.247048 SER_134 63.047237 ASN_135 31.403708 SER_136
97.999619 SER_137 28.505201 GLN_138 102.845520 PRO_139 49.691917
SER_140 9.423104 PRO_141 25.724171 PRO_142 80.706665 SER_143
105.318176 LEU_144 20.154398 ILE_145 41.288322 PRO_146 10.462679
GLY_147 19.803421 PRO_148 18.130360 GLY_149 47.391853 ASN_150
60.248917 THR_151 87.887985 VAL_152 13.870322 THR_153 74.664734
ALA_154 45.251106 ILE_155 2.686934 LEU_156 28.720940 ASP_157
110.081253 ARG_158 31.228874 MET_159 1.612160 GLY_160 38.223858
ASP_161 46.293152 ALA_162 9.877204 GLY_163 34.267326 PHE_164
11.057570 SER_165 51.158882 PRO_166 62.767738 ASP_167 75.164917
GLU_168 43.334976 VAL_169 6.365355 VAL_170 2.955627 ASP_171
7.004863 LEU_172 1.880854 LEU_173 3.197691 ALA_174 0.000000 ALA_175
1.074774 HIS_176 0.502189 SER_177 0.806080 LEU_178 3.197691 ALA_179
3.337480 SER_180 0.466991 GLN_181 2.122917 GLU_182 40.996552
GLY_183 62.098671 LEU_184 23.954853 ASN_185 15.918136 SER_186
95.185318 ALA_187 59.075272 ILE_188 27.675419 PHE_189 102.799423
ARG_190 55.265549 SER_191 6.986028 PRO_192 2.686934 LEU_193
12.321225 ASP_194 2.127163 SER_195 33.556419 THR_196 33.049286
PRO_197 20.874798 GLN_198 65.729698 VAL_199 31.705818 PHE_200
4.753195 ASP_201 13.744506 THR_202 1.612160 GLN_203 16.081930
PHE_204 2.581340 TYR_205 1.880854 ILE_206 9.356181 GLU_207 0.735684
THR_208 10.685907 LEU_209 9.672962 LEU_210 2.955627 LYS_211
77.176834 GLY_212 40.968609 THR_213 78.718216 THR_214 21.738384
GLN_215 77.622299 PRO_216 25.441587 GLY_217 8.320850 PRO_218
96.972305 SER_219 64.627823 LEU_220 85.732414 GLY_221 27.361111
PHE_222 134.620178 ALA_223 3.873014 GLU_224 12.141763 GLU_225
65.129868 LEU_226 76.105843 SER_227 0.268693 PRO_228 7.017754
PHE_229 0.000000 PRO_230 47.827423 GLY_231 23.790522 GLU_232
6.643466 PHE_233 6.713862 ARG_234 18.012030 MET_235 4.598188
ARG_236 91.415581 SER_237 1.982125 ASP_238 6.246871 ALA_239
12.897283 LEU_240 76.820526 LEU_241 3.224321 ALA_242 1.400973
ARG_243 77.207176 ASP_244 36.207306 SER_245 104.023796 ARG_246
121.852341 THR_247 2.955627 ALA_248 4.810700 CYS_249 47.331306
ARG_250 62.062778 TRP_251 2.418241 GLN_252 5.554953 SER_253
38.284832 MET_254 1.124224 THR_255 0.000000 SER_256 53.758987
SER_257 37.276134 ASN_258 44.381340 GLU_259 149.565140 VAL_260
57.500389 MET_261 2.679314 GLY_262 10.175152 GLN_263 107.458916
ARG_264 36.402130 TYR_265 0.233495 ARG_266 91.179619 ALA_267
53.708500 ALA_268 6.504294 MET_269 17.122011 ALA_270 22.455158
LYS_271 73.386177 MET_272 3.959508 SER_273 15.043281 VAL_274
23.887930 LEU_275 17.196379 GLY_276 44.362202 PHE_277 68.062485
ASP_278 94.902039 ARG_279 113.549011 ASN_280 134.886017 ALA_281
72.340973 LEU_282 26.692348 THR_283 27.696728 ASP_284 72.214157
CYS_285 0.000000 SER_286 28.209335 ASP_287 64.560753 VAL_288
7.040061 ILE_289 8.665112 PRO_290 48.682365 SER_291 86.141670
ALA_292 29.031240 VAL_293 84.432014 SER_294 85.944153 ASN_295
49.017288 ASN_296 133.459198 ALA_297 57.283794 ALA_298 65.233749
PRO_299 24.751518 VAL_300 45.409184 ILE_301 8.060802 PRO_302
14.742939 GLY_303 16.589832 GLY_304 34.238071 LEU_305 24.719791
THR_306 49.356300 VAL_307 71.491821 ASP_308 130.906174 ASP_309
31.733070 ILE_310 19.581894 GLU_311 81.414574 VAL_312 94.769890
SER_313 39.688896 CYS_314 9.998511 PRO_315 120.328018 SER_316
95.364319 GLU_317 65.560959 PRO_318 100.254364 PHE_319 46.284115
PRO_320 31.328060 GLU_321 177.602249 ILE_322 33.449741 ALA_323
46.892982 THR_324 79.976471 ALA_325 36.423820 SER_326 124.467422
GLY_327 28.219524 PRO_328 107.553696 LEU_329 86.789825 PRO_330
34.287163 SER_331 75.764053 LEU_332 32.840569 ALA_333 61.516434
PRO_334 82.389992 ALA_335 6.246871 PRO_336 56.750813 HEM_337
60.435017 CA_338 2.078997 CA_339 0.000000 NAG_340 141.534668
NAG_341 186.311371 Subset REST: restmole.list Subset REST: ARP: 9,
69-70, 125, 127, 133, 299-301, 334-336 restatom.list Subset REST:
ARP: SER 9: N, CA, C, O, CB, OG ARP: GLY 69: N, CA, C, O ARP: GLN
70: N, CA, C, O, CB, CG, CD, OE1, NE2 ARP: GLY 125: N, CA, C, O
ARP: SER 127: N, CA, C, O, CB, OG ARP: PRO 133: N, CA, CD, C, O,
CB, CG ARP: SER 299: N, CA, C, O, CB, OG ARP: ALA 300: N, CA, C, O,
CB ARP: VAL 301: N, CA, C, O, CB, CG1, CG2 ARP: SER 334: N, CA, C,
O, CB, OG ARP: GLY 335: N, CA, C, O ARP: PRO 336: N, CA, CD, C, O,
CB, CG Subset SUB5B: sub5bmole.list Subset SUB5B: ARP: 10-11, 34,
38, 65-68, 71-72, 120-121, 123-124, 128-132, 134, 270, 274, ARP:
297-298, 302-303, 311-312, 332-333, 337-338 sub5batom.list Subset
SUB5B: ARP: VAL 10: N, CA, C, O, CB, CG1, CG2 ARP: THR 11: N, CA,
C, O, CB, OG1, CG2 ARP: GLN 34: N, CA, C, O, CB, CG, CD, OE1, NE2
ARP: TYR 38: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH ARP:
LEU 65: N, CA, C, O, CB, CG, CD1, CD2 ARP: THR 66: N, CA, C, O, CB,
OG1, CG2 ARP: ALA 67: N, CA, C, O, CB ARP: ALA 68: N, CA, C, O, CB
ARP: PHE 71: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ ARP: GLY
72: N, CA, C, O ARP: PHE 120: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ ARP: ALA 121: N, CA, C, O, CB ARP: ALA 123: N, CA, C, O, CB
ARP: VAL 124: N, CA, C, O, CB, CG1, CG2 ARP: ASN 128: N, CA, C, O,
CB, CG, OD1, ND2 ARP: CYS 129: N, CA, C, O, CB, SG ARP: PRO 130: N,
CA, CD, C, O, CB, CG ARP: GLY 131: N, CA, C, O ARP: SER 132: N, CA,
C, O, CB, OG ARP: ARG 134: N, CA, C, O, CB, CG, CD, NE, CZ, NH1,
NH2 ARP: GLY 270: N, CA, C, O ARP: ARG 274: N, CA, C, O, CB, CG,
CD, NE, CZ, NH1, NH2 ARP: ILE 297: N, CA, C, O, CB, CG1, CG2, CD1
ARP: PRO 298: N, CA, CD, C, O, CB, CG ARP: SER 302: N, CA, C, O,
CB, OG ARP: ASN 303: N, CA, C, O, CB, CG, OD1, ND2 ARP: GLY 311: N,
CA, C, O ARP: GLY 312: N, CA, C, O ARP: THR 332: N, CA, C, O, CB,
OG1, CG2 ARP: ALA 333: N, CA, C, O, CB ARP: LEU 337: N, CA, C, O,
CB, CG, CD1, CD2 ARP: PRO 338: N, CA, CD, C, O, CB, CG Subset
ACTSITE: actsitemole.list Subset ACTSITE: ARP: 44-61, 75-77, 79-80,
87-88, 90-96, 99, 118, 122, 126, 135, 148-149, 152-158, ARP:
163-164, 167, 176-194, 197-205, 207-209, 211-213, 216, 230-231,
241, ARP: 243-246, 249, 259, 273, 277, 280, 343-347H
actsiteatom.list Subset ACTSITE: ARP: GLU 44: N, CA, C, O, CB, CG,
CD, OE1, OE2 ARP: SER 45: N, CA, C, O, CB, OG ARP: PRO 46: N, CA,
CD, C, O, CB, CG ARP: VAL 47: N, CA, C, O, CB, CG1, CG2 ARP: ARG
48: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 ARP: LYS 49: N, CA,
C, O, CB, CG, CD, CE, NZ ARP: ILE 50: N, CA, C, O, CB, CG1, CG2,
CD1 ARP: LEU 51: N, CA, C, O, CB, CG, CD1, CD2 ARP: ARG 52: N, CA,
C, O, CB, CG, CD, NE, CZ, NH1, NH2 ARP: ILE 53: N, CA, C, O, CB,
CG1, CG2, CD1 ARP: VAL 54: N, CA, C, O, CB, CG1, CG2 ARP: PHE 55:
N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ ARP: HIS 56: N, CA, C,
O, CB, CG, ND1, CD2, CE1, NE2 ARP: ASP 57: N, CA, C, O, CB, CG,
OD1, OD2 ARP: ALA 58: N, CA, C, O, CB ARP: ILE 59: N, CA, C, O, CB,
CG1, CG2, CD1 ARP: GLY 60: N, CA, C, O ARP: PHE 61: N, CA, C, O,
CB, CG, CD1, CD2, CE1, CE2, CZ ARP: GLY 75: N, CA, C, O ARP: ALA
76: N, CA, C, O, CB ARP: ASP 77: N, CA, C, O, CB, CG, OD1, OD2 ARP:
SER 79: N, CA, C, O, CB, OG ARP: ILE 80: N, CA, C, O, CB, CG1, CG2,
CD1 ARP: GLU 87: N, CA, C, O, CB, CG, CD, OE1, OE2 ARP: LEU 88: N,
CA, C, O, CB, CG, CD1, CD2 ARP: PHE 90: N, CA, C, O, CB, CG, CD1,
CD2, CE1, CE2, CZ ARP: PRO 91: N, CA, CD, C, O, CB, CG ARP: ALA 92:
N, CA, C, O, CB ARP: ASN 93: N, CA, C, O, CB, CG, OD1, ND2 ARP: GLY
94: N, CA, C, O ARP: GLY 95: N, CA, C, O ARP: LEU 96: N, CA, C, O,
CB, CG, CD1, CD2 ARP: THR 99: N, CA, C, O, CB, OG1, CG2 ARP: ILE
118: N, CA, C, O, CB, CG1, CG2, CD1 ARP: THR 122: N, CA, C, O, CB,
OG1, CG2 ARP: MET 126: N, CA, C, O, CB, CG, SD, CE ARP: LEU 135: N,
CA, C, O, CB, CG, CD1, CD2 ARP: SER 148: N, CA, C, O, CB, OG ARP:
PRO 149: N, CA, CD, C, O, CB, CG ARP: LEU 152: N, CA, C, O, CB, CG,
CD1, CD2 ARP: ILE 153: N, CA, C, O, CB, CG1, CG2, CD1 ARP: PRO 154:
N, CA, CD, C, O, CB, CG ARP: GLY 155: N, CA, C, O ARP: PRO 156: N,
CA, CD, C, O, CB, CG ARP: GLY 157: N, CA, C, O ARP: ASN 158: N, CA,
C, O, CB, CG, OD1, ND2 ARP: ILE 163: N, CA, C, O, CB, CG1, CG2, CD1
ARP: LEU 164: N, CA, C, O, CB, CG, CD1, CD2 ARP: MET 167: N, CA, C,
O, CB, CG, SD, CE ARP: GLU 176: N, CA, C, O, CB, CG, CD, OE1, OE2
ARP: VAL 177: N, CA, C, O, CB, CG1, CG2 ARP: VAL 178: N, CA, C, O,
CB, CG1, CG2 ARP: ASP 179: N, CA, C, O, CB, CG, OD1, OD2 ARP: LEU
180: N, CA, C, O, CB, CG, CD1, CD2 ARP: LEU 181: N, CA, C, O, CB,
CG, CD1, CD2 ARP: ALA 182: N, CA, C, O, CB ARP: ALA 183: N, CA, C,
O, CB ARP: HIS 184: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2 ARP:
SER 185: N, CA, C, O, CB, OG ARP: LEU 186: N, CA, C, O, CB, CG,
CD1, CD2 ARP: ALA 187: N, CA, C, O, CB ARP: SER 188: N, CA, C, O,
CB, OG ARP: GLN 189: N, CA, C, O, CB, CG, CD, OE1, NE2 ARP: GLU
190: N, CA, C, O, CB, CG, CD, OE1, OE2 ARP: GLY 191: N, CA, C, O
ARP: LEU 192: N, CA, C, O, CB, CG, CD1, CD2 ARP: ASN 193: N, CA, C,
O, CB, CG, OD1, ND2 ARP: SER 194: N, CA, C, O, CB, OG ARP: PHE 197:
N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ ARP: ARG 198: N, CA, C,
O, CB, CG, CD, NE, CZ, NH1, NH2 ARP: SER 199: N, CA, C, O, CB, OG
ARP: PRO 200: N, CA, CD, C, O, CB, CG ARP: LEU 201: N, CA, C, O,
CB, CG, CD1, CD2 ARP: ASP 202: N, CA, C, O, CB, CG, OD1, OD2 ARP:
SER 203: N, CA, C, O, CB, OG ARP: THR 204: N, CA, C, O, CB, OG1,
CG2 ARP: PRO 205: N, CA, CD, C, O, CB, CG ARP: VAL 207: N, CA, C,
O, CB, CG1, CG2 ARP: PHE 208: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ ARP: ASP 209: N, CA, C, O, CB, CG, OD1, OD2 ARP: GLN 211:
N, CA, C, O, CB, CG, CD, OE1, NE2 ARP: PHE 212: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ ARP: TYR 213: N, CA, C, O, CB, CG, CD1,
CD2, CE1, CE2, CZ, OH ARP: THR 216: N, CA, C, O, CB, OG1, CG2 ARP:
PHE 230: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ ARP: ALA 231:
N, CA, C, O, CB ARP: PHE 241: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ ARP: MET 243: N, CA, C, O, CB, CG, SD, CE ARP: ARG 244: N,
CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 ARP: SER 245: N, CA, C, O,
CB, OG ARP: ASP 246: N, CA, C, O, CB, CG, OD1, OD2 ARP: LEU 249: N,
CA, C, O, CB, CG, CD1, CD2 ARP: TRP 259: N, CA, C, O, CB, CG, CD1,
CD2, NE1, CE2, CE3, CZ2, CZ3, CH2 ARP: TYR 273: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ, OH ARP: MET 277: N, CA, C, O, CB, CG,
SD, CE ARP: MET 280: N, CA, C, O, CB, CG, SD, CE ARP: ALA 343: N,
CA, C, O, CB ARP: PRO 344: N, CA, CD, C, O, OXT, CB, CG ARP: HEM
345H: FE, NA, NB, NC, ND, CHA, CHB, CHC, CHD, C1A, C2A, C3A, C4A,
CMA, CAA, CBA, CGA ARP: HEM 345H: O1A, O2A, C1B, C2B, C3B, C4B,
CMB, CAB, CBB, C1C, C2C, C3C, C4C, CMC, CAC, CBC ARP: HEM 345H:
C1D, C2D, C3D, C4D, CMD, CAD, CBD, CGD, O1D, O2D ARP: CA 346H: CA
ARP: CA 347H: CA Subset RESTx: restxmole.list Subset RESTX
NEWMODEL: 9, 334-336 restxatom.list Subset RESTX: NEWMODEL: SER 9:
N, CA, C, O, CB, OG NEWMODEL: SER 334: N, CA, C, O, CB, OG
NEWMODEL: GLY 335: N, CA, C, O NEWMODEL: PRO 336: N, CA, CD, C, O,
CB, CG
Example 5
[0715] Activation of mPEG 15,000 with N-succinimidyl Carbonate
[0716] mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) 20%
was distilled off at normal pressure to dry the reactants
azeotropically. Dichloromethane (dry 1 ml/g mPEG) was added when
the solution was cooled to 30.degree. C. and phosgene in toluene
(1.93 M 5 mole/mole mPEG) was added and mixture stirred at room
temperature overnight. The mixture was evaporated to dryness and
the desired product was obtained as waxy lumps.
[0717] After evaporation dichloromethane and toluene (1:2, dry 3
ml/g mPEG) was added to re-dissolve the white solid. N-Hydroxy
succinimide (2 mole/mole mPEG) was added as a solid and then
triethylamine (1.1 mole/mole mPEG). The mixture was stirred for 3
hours, initially unclear, then clear and ending with a small
precipitate. The mixture was evaporated to dryness and
recrystallized from ethyl acetate (10 ml) with warm filtration to
remove salts and insoluble traces. The blank liquid was left for
slow cooling at ambient temperature for 16 hours and then in the
refrigerator overnight. The white precipitate was filtered and
washed with a little cold ethyl acetate and dried to yield 98%
(w/w). NMR Indicating 80-90% activation and 5 o/oo (w/w)
HNEt.sub.3Cl. .sup.1H-NMR for mPEG 15,000 (CDCl.sub.3) d 1.42 t
(I=4.8 CH.sub.3 i HNEt.sub.3Cl), 2.84 s (I=3.7 succinimide), 3.10
dq (I=3.4 CH.sub.2 i HNEt.sub.3Cl), 3.38 s (I=2.7 CH.sub.3 i OMe),
3.40* dd (I=4.5 o/oo, .sup.13C satellite), 3.64 bs (I=1364 main
peak), 3.89* dd (I=4.8 o/oo, .sup.13C satellite), 4.47 dd (I=1.8,
CH.sub.2 in PEG). No change was seen after storage in a desiccator
at 22.degree. C. for 4 months.
Example 6
[0718] Activation of mPEG 5,000 with N-succinimidyl Carbonate
[0719] Activation of mPEG 5,000 with N-succinimidyl carbonate was
performed as described in Example 5.
Example 7
[0720] Construction and Expression of PD498 Variants:
[0721] PD498 site-directed variants were constructed using the
"maxi-oligonucleotide-PCR" method described by Sarkar et al., 1990,
BioTechniques, 8, 404-407.
[0722] The template plasmid was shuttle vector pPD498 or an
analogue of this containing a variant of the PD498 protease
gene.
[0723] The following PD498 variants were constructed, expressed and
purified.
[0724] A: R28K
[0725] B: R62K
[0726] C: R169K
[0727] D: R28K.sup.+ R62K
[0728] E: R28K.sup.+ R169K
[0729] F: R62K.sup.+ R169K
[0730] G: R28K.sup.+ R69K.sup.+ R169K.
[0731] Construction of Variants
[0732] For introduction of the R28K substitution a synthetic
oligonucleotide having the sequence: GGG ATG TAA CCA AGG GAA GCA
GCA CTC AAA CG (SEQ ID NO: 7) was used.
[0733] A PCR fragment of 769 bp was ligated into the pPD498 plasmid
prepared by Bst E II and Bgl II digestion. Positive variants were
recognized by Styl digestion and verified by DNA sequencing of the
total 769 bp insert.
[0734] For introduction of the R62K substitution a synthetic
oligonucleotide having the sequence: CGA CTT TAT CGA TAA GGA CAA
TAA CCC (SEQ ID NO: 8) was used.
[0735] A PCR fragment of 769 bp was ligated into the pPD498 plasmid
prepared by Bst E II and Bgl II digestion. Positive variants were
recognized by ClaI digestion and verified by DNA sequencing of the
total 769 bp insert.
[0736] For introduction of the R169K substitution a synthetic
oligonucleotide having the sequence: CAA TGT ATC CAA AAC GTT CCA
ACC AGC (SEQ ID NO: 9) was used.
[0737] A PCR fragment of 769 bp was ligated into the pPD498 plasmid
prepared by Bst E II and Bgl II digestion. Positive variants were
recognized by the absence of an Rsa I restriction site and verified
by DNA sequencing of the total 769 bp insert.
[0738] For simultaneous introduction of the R28K and the R62K
substitutions, synthetic oligonucleotides having the sequence GGG
ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) and the
sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) were
used simultaneously. A PCR fragment of 769 bp was ligated into the
pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive
variants were recognized by Styl and ClaI digestion and verified by
DNA sequencing of the total 769 bp insert.
[0739] For simultaneous introduction of the R28K and the R169K
substitutions, synthetic oligonucleotides having the sequence GGG
ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) and the
sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were
used simultaneously. A PCR fragment of 769 bp was ligated into the
pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive
variants were recognized by Styl digestion and absence of an Rsa I
site. The variant was verified by DNA sequencing of the total 769
bp insert.
[0740] For simultaneous ntroduction of the R62K and the R169K
substitutions, synthetic oligonucleotides having the sequence CGA
CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) and the sequence CAA
TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were used
simultaneously. A PCR fragment of 769 bp was ligated into the
pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive
variants were recognized by ClaI digestion and absence of an Rsa I
site. The variant was verified by DNA sequencing of the total 769
bp insert.
[0741] For simultaneous introduction of the R28K, the R62K and the
R169K substitutions, synthetic oligonucleotides having the sequence
GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7), the
sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) and the
sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were
used simultaneously. A PCR fragment of 769 bp was ligated into the
pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive
variants were recognized by Styl and ClaI digestion and absence of
an Rsa I site. The variant was verified by DNA sequencing of the
total 769 bp insert.
[0742] Fermentation, Expression and Purification of PD498
Variants
[0743] Vectors hosting the above mentioned PD498 variants were
purified from E. coli cultures and transformed into B. subtilis in
which organism the variants were fermented, expressed and purified
as described in the "Materials and Methods" section above.
Example 8
[0744] Conjugation of Triple Substituted PD498 Variant with
Activated mPEG 5,000
[0745] 200 mg of triple substituted PD498 variant (i.e. the
R28K.sup.+ R62K.sup.+ R169K substituted variant) was incubated in
50 mm NaBorate, pH 10, with 1.8 g of activated mPEG 5,000 with
N-succinimidyl carbonate (prepared according to Example 2), in a
final volume of 20 ml. The reaction was carried out at ambient
temperature using magnetic stirring. Reaction time was 1 hour. The
reaction was stopped by adding DMG buffer to a final concentration
of 5 mM dimethyl glutarate, 1 mM CaCl.sub.2 and 50 mM borate, pH
5.0.
[0746] The molecule weight of the obtained derivative was
approximately 120 kDa, corresponding to about 16 moles of mPEG
attached per mole enzyme.
[0747] Compared to the parent enzyme, residual activity was close
to 100% towards peptide substrate
(succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide).
Example 9
[0748] Allergenicity Trials of PD498 Variant-SPEG 5,000 in Guinea
Pigs
[0749] Dunkin Hartley guinea pigs are stimulated with 1.0 microgram
PD498-SPEG 5,000 and 1.0 microgram modified variant PD498-SPEG
5,000 by intratracheal installation.
[0750] Sera from immunized Dunkin Hartley guinea pigs are tested
during the trial period in a specific IgG.sub.1 ELISA (described
above) to elucidate whether the molecules could activate the immune
response system giving rise to a specific IgG.sub.1 response
indicating an allergenic response.
[0751] The IgG.sub.1 levels of Dunkin Hartley guinea pigs during
the trial period of 10 weeks are observed.
Example 10
[0752] Suitable Substitutions in Humicola lanuginosa Lipase for
Addition of Amino Attachment Groups (--NH.sub.2)
[0753] The 3D structure of Humicola lanuginosa lipase (SEQ ID NO:
6) is available in Brookhaven Databank as ltib.pdb. The lipase
consists of 269 amino acids.
[0754] The procedure described in Example 1 was followed. The
sequence of H. lanuginosa lipase is shown below in the table
listing solvent accessibility data for H. lanuginosa lipase. H.
lanuginosa residue numbering is used (1-269), and the active site
residues (functional site) are S146, S201 and H258. The synonym TIB
is used for H. lanuginosa lipase.
[0755] The commands performed in Insight (BIOSYM) are shown in the
command files makeKzone.bcl and makeKzone2.bcl below:
[0756] Conservative Substitutions:
[0757] makeKzone.bcl
[0758] 1 Delete Subset *
[0759] 2 Color Molecule Atoms * Specified Specification
255,0,255
[0760] 3 Zone Subset LYS :lys:NZ Static monomer/residue 10
Color_Subset 255,255,0
[0761] 4 Zone Subset NTERM :1:N Static monomer/residue 10
Color_Subset 255,255,0
[0762] 5 #NOTE: editnextline ACTSITE residues according to the
protein
[0763] 6 Zone Subset ACTSITE :146,201,258 Static monomer/residue 8
Color_Subset 255,255,0
[0764] 7 Combine Subset ALLZONE Union LYS NTERM
[0765] 8 Combine Subset ALLZONE Union ALLZONE ACTSITE
[0766] 9 #NOTE: editnextline object name according to the
protein
[0767] 10 Combine Subset REST Difference TIB ALLZONE
[0768] 11 List Subset REST Atom Output_File restatom.list
[0769] 12 List Subset REST monomer/residue Output_File
restmole.list
[0770] 13 Color Molecule Atoms ACTSITE Specified Specification
255,0,0
[0771] 14 List Subset ACTSITE Atom Output_File actsiteatom.list
[0772] 15 List Subset ACTSITE monomer/residue Output_File
actsitemole.list
[0773] 16 #
[0774] 17 Zone Subset REST5A REST Static Monomer/Residue
5-Color_Subset
[0775] 18 Combine Subset SUB5A Difference REST5A ACTSITE
[0776] 19 Combine Subset SUB5B Difference SUB5A REST
[0777] 20 Color Molecule Atoms SUB5B Specified Specification
255,255,255
[0778] 21 List Subset SUB5B Atom Output_File sub5batom.list
[0779] 22 List Subset SUB5B monomer/residue Output_File
sub5bmole.list
[0780] 23 #Now identify sites for lys->arg substitutions and
continue with makezone2.bcl
[0781] 24 #Use grep command to identify ARG in restatom.list,
sub5batom.list & accsiteatom.list.
[0782] Comments:
[0783] In this case of H. lanuginosa (=TIB), REST contains the
arginines Arg133, Arg139, Arg160, Arg179 and Arg 209, and SUB5B
contains Arg118 and R125.
[0784] These residues are all solvent exposed. The substitutions
R133K, R139K, R160K, R179K, R209K, R118K and R125K are identified
in TIB as sites for mutagenesis within the scope of this invention.
The residues are substituted below in section 2, and further
analysis done. The subset ACTSITE contains no lysines.
[0785] Non-Conservative Substitutions:
[0786] makeKzone2.bcl
[0787] 1 #sourcefile makezone2.bcl Claus von der Osten 961128
[0788] 2 #
[0789] 3 #having scanned lists (grep arg command) and identified
sites for lys->arg substitutions
[0790] 4 #NOTE: editnextline object name according to protein
[0791] 5 Copy Object -To_Clipboard -Displace TIB newmodel
[0792] 6 Biopolymer
[0793] 7 #NOTE: editnextline object name according to protein
[0794] 8 Blank Object On TIB
[0795] 9 #NOTE: editnextlines with lys->arg positions
[0796] 10 Replace Residue newmodel:118 lys L
[0797] 11 Replace Residue newmodel:125 lys L
[0798] 12 Replace Residue newmodel:133 lys L
[0799] 13 Replace Residue newmodel:139 lys L
[0800] 14 Replace Residue newmodel:160 lys L
[0801] 15 Replace Residue newmodel:179 lys L
[0802] 16 Replace Residue newmodel:209 lys L
[0803] 17 #
[0804] 18 #Now repeat analysis done prior to arg->lys, now
including introduced lysines
[0805] 19 Color Molecule Atoms newmodel Specified Specification
255,0,255
[0806] 20 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue
10 Color_Subset 255,255,0
[0807] 21 Zone Subset NTERMx newmodel:l:N Static monomer/residue 10
Color_Subset 255,255,0
[0808] 22 #NOTE: editnextline ACTSITEx residues according to the
protein
[0809] 23 Zone Subset ACTSITEx newmodel:146,201,258 Static
monomer/residue 8 Color_Subset 255,255,0
[0810] 24 Combine Subset ALLZONEx Union LYSx NTERMx
[0811] 25 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
[0812] 26 Combine Subset RESTx Difference newmodel ALLZONEx
[0813] 27 List Subset RESTx Atom Output_File restxatom.list
[0814] 28 List Subset RESTx monomer/residue Output_File
restxmole.list
[0815] 29 #
[0816] 30 Color Molecule Atoms ACTSITEx Specified Specification
255,0,0
[0817] 31 List Subset ACTSITEx Atom Output_File
actsitexatom.list
[0818] 32 List Subset ACTSITEx monomer/residue Output_File
actsitexmole.list
[0819] 33 #
[0820] 34 #read restxatom.list or restxmole.list to identify sites
for (not_arg)->lys subst. if needed.
[0821] Comments:
[0822] Of the residues in RESTx, the following are >5% exposed
(see lists below): 18, 31-33, 36, 38, 40, 48, 50, 56-62, 64, 78,
88, 91-93, 104-106, 120, 136, 225, 227-229, 250, 262, 268. Of these
three are cysteines involved in disulfide bridge formation, and
consequently for structural reasons excluded from the residues to
be mutated. The following mutations are proposed in H. lanuginosa
lipase (TIB): A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K,
D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K,
L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K,
P250K, F262K.
[0823] Relevant Data for Example 10:
11 # TIBNOH2O # residue area GLU_1 110.792610 VAL_2 18.002457 SER_3
53.019516 GLN_4 85.770164 ASP_5 107.565826 LEU_6 33.022659 PHE_7
34.392754 ASN_8 84.855331 GLN_9 39.175591 PHE_10 2.149547 ASN_11
40.544380 LEU_12 27.648788 PHE_13 2.418241 ALA_14 4.625293 GLN_15
28.202387 TYR_16 0.969180 SER_17 0.000000 ALA_18 7.008336 ALA_19
0.000000 ALA_20 0.000000 TYR_21 6.947358 CYS_22 8.060802 GLY_23
32.147034 LYS_24 168.890747 ASN_25 8.014721 ASN_26 11.815564 ASP_27
92.263428 ALA_28 18.206699 PRO_29 83.188431 ALA_30 69.428421 GLY_31
50.693439 THR_32 52.171135 ASN_33 111.230743 ILE_34 2.801945 THR_35
82.130569 CYS_36 17.269245 THR_37 96.731941 GLY_38 77.870995 ASN_39
123.051003 ALA_40 27.985256 CYS_41 0.752820 PRO_42 46.258949 GLU_43
69.773987 VAL_44 0.735684 GLU_45 77.169510 LYS_46 141.213562 ALA_47
10.249716 ASP_48 109.913902 ALA_49 2.602721 THR_50 32.012184 PHE_51
8.255627 LEU_52 60.093613 TYR_53 77.877937 SER_54 26.980494 PHE_55
10.747735 GLU_56 112.689758 ASP_57 92.064278 SER_58 32.990780
GLY_59 53.371807 VAL_60 83.563644 GLY_61 69.625633 ASP_62 75.520988
VAL_63 4.030401 THR_64 8.652839 GLY_65 0.000000 PHE_66 0.268693
LEU_67 11.822510 ALA_68 0.537387 LEU_69 30.243870 ASP_70 0.000000
ASN_71 84.101044 THR_72 89.271126 ASN_73 70.742401 LYS_74 98.319168
LEU_75 8.329495 ILE_76 5.197878 VAL_77 0.806080 LEU_78 5.293978
SER_79 0.000000 PHE_80 2.079151 ARG_81 41.085312 GLY_82 1.471369
SER_83 43.794014 ARG_84 100.261627 SER_85 70.607552 ILE_86
59.696865 GLU_87 136.510773 ASN_88 119.376373 TRP_89 102.851227
ILE_90 78.068588 GLY_91 60.783607 ASN_92 45.769428 LEU_93
134.228363 ASN_94 101.810959 PHE_95 41.212212 ASP_96 79.645950
LEU_97 25.281572 LYS_98 88.840263 GLU_99 132.377090 ILE_100
9.135575 ASN_101 63.444527 ASP_102 88.652847 ILE_103 33.470661
CYS_104 11.553816 SER_105 99.461174 GLY_106 40.325161 CYS_107
4.433561 ARG_108 97.450104 GLY_109 1.343467 HIS_110 4.652464
ASP_111 37.023655 GLY_112 29.930408 PHE_113 14.976435 THR_114
10.430954 SER_115 40.606895 SER_116 13.462922 TRP_117 10.747735
ARG_118 114.364281 SER_119 46.880249 VAL_120 13.434669 ALA_121
18.258261 ASP_122 110.753098 THR_123 69.641922 LEU_124 17.090784
ARG_125 73.929977 GLN_126 101.320190 LYS_127 84.450241 VAL_128
6.448641 GLU_129 47.700993 ASP_130 75.529091 ALA_131 11.340775
VAL_132 27.896025 ARG_133 153.136490 GLU_134 132.140594 HIS_135
54.553406 PRO_136 97.386963 ASP_137 22.653191 TYR_138 35.392658
ARG_139 74.321243 VAL_140 10.173222 VAL_141 0.233495 PHE_142
3.224321 THR_143 0.000000 GLY_144 0.000000 HIS_145 4.514527 SER_146
15.749787 LEU_147 40.709171 GLY_148 0.000000 GLY_149 0.000000
ALA_150 0.537387 LEU_151 22.838938 ALA_152 0.268693 THR_153
18.078798 VAL_154 7.254722 ALA_155 0.000000 GLY_156 0.000000
ALA_157 15.140230 ASP_158 41.645477 LEU_159 6.144750 ARG_160
41.939716 GLY_161 68.978180 ASN_162 68.243805 GLY_163 79.181274
TYR_164 36.190247 ASP_165 103.068283 ILE_166 0.000000 ASP_167
24.326443 VAL_168 4.299094 PHE_169 0.466991 SER_170 3.339332
TYR_171 0.000000 GLY_172 0.000000 ALA_173 12.674671 PRO_174
13.117888 ARG_175 10.004488 VAL_176 21.422220 GLY_177 2.680759
ASN_178 21.018063 ARG_179 110.282166 ALA_180 33.210381 PHE_181
4.567788 ALA_182 3.897251 GLU_183 76.354004 PHE_184 71.225983
LEU_185 24.985012 THR_186 47.023815 VAL_187 98.244606 GLN_188
54.152954 THR_189 88.660645 GLY_190 24.792120 GLY_191 10.726818
THR_192 45.458744 LEU_193 16.633211 TYR_194 34.829491 ARG_195
29.030851 ILE_196 1.973557 THR_197 3.493014 HIS_198 1.532270
THR_199 34.785877 ASN_200 39.789238 ASP_201 0.000000 ILE_202
31.168434 VAL_203 29.521076 PRO_204 3.515322 ARG_205 44.882454
LEU_206 51.051746 PRO_207 12.575329 PRO_208 43.259636 ARG_209
113.700233 GLU_210 154.628540 PHE_211 112.505188 GLY_212 30.084938
TYR_213 3.268936 SER_214 12.471436 HIS_215 23.354481 SER_216
16.406200 SER_217 14.665598 PRO_218 17.240993 GLU_219 13.145291
TYR_220 18.718306 TRP_221 39.229233 ILE_222 5.105175 LYS_223
120.739983 SER_224 15.407301 GLY_225 29.306646 THR_226 66.806862
LEU_227 122.682808 VAL_228 60.923004 PRO_229 104.620377 VAL_230
23.398251 THR_231 63.372971 ARG_232 80.357857 ASN_233 89.255066
ASP_234 43.011250 ILE_235 2.114349 VAL_236 45.140491 LYS_237
105.651306 ILE_238 24.671705 GLU_239 116.891907 GLY_240 31.965794
ILE_241 46.278099 ASP_242 28.963699 ALA_243 25.158146 THR_244
98.351440 GLY_245 43.842186 GLY_246 0.700486 ASN_247 3.926274
ASN_248 51.047890 GLN_249 66.699188 PRO_250 132.414047 ASN_251
70.213730 ILE_252 141.498062 PRO_253 59.089233 ASP_254 59.010895
ILE_255 63.298943 PRO_256 78.608688 ALA_257 0.806080 HIS_258
3.761708 LEU_259 50.747856 TRP_260 35.229710 TYR_261 5.440791
PHE_262 36.457939 GLY_263 22.071375 LEU_264 109.148178 ILE_265
2.418241 GLY_266 17.730062 THR_267 68.217873 CYS_268 15.418195
LEU_269 165.990997 Subset REST: restmole.list Subset REST: TIB: 5,
8-9, 13-14, 16, 18-20, 31-34, 36, 38, 40, 48-50, 56-66, 68, 76-79,
88, 91-93, TIB: 100-107, 116-117, 119-121, 132-134, 136, 139-142,
154-169, 177-185, TIB: 187, 189-191, 207-212, 214-216, 225,
227-229, 241-244, 250, 262, 268 restatom.list Subset REST: TIB: ASP
5: N, CA, C, O, CB, CG, OD1, OD2 TIB: ASN 8: N, CA, C, O, CB, CG,
OD1, ND2 TIB: GLN 9: N, CA, C, O, CB, CG, CD, OE1, NE2 TIB: PHE 13:
N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: ALA 14: N, CA, C,
O, CB TIB: TYR 16: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
TIB: ALA 18: N, CA, C, O, CB TIB: ALA 19: N, CA, C, O, CB TIB: ALA
20: N, CA, C, O, CB TIB: GLY 31: N, CA, C, O TIB: THR 32: N, CA, C,
O, CB, OG1, CG2 TIB: ASN 33: N, CA, C, O, CB, CG, OD1, ND2 TIB: ILE
34: N, CA, C, O, CB, CG1, CG2, CD1 TIB: CYS 36: N, CA, C, O, CB, SG
TIB: GLY 38: N, CA, C, O TIB: ALA 40: N, CA, C, O, CB TIB: ASP 48:
N, CA, C, O, CB, CG, OD1, OD2 TIB: ALA 49: N, CA, C, O, CB TIB: THR
50: N, CA, C, O, CB, OG1, CG2 TIB: GLU 56: N, CA, C, O, CB, CG, CD,
OE1, OE2 TIB: ASP 57: N, CA, C, O, CB, CG, OD1, OD2 TIB: SER 58: N,
CA, C, O, CB, OG TIB: GLY 59: N, CA, C, O TIB: VAL 60: N, CA, C, O,
CB, CG1, CG2 TIB: GLY 61: N, CA, C, O TIB: ASP 62: N, CA, C, O, CB,
CG, OD1, OD2 TIB: VAL 63: N, CA, C, O, CB, CG1, CG2 TIB: THR 64: N,
CA, C, O, CB, OG1, CG2 TIB: GLY 65: N, CA, C, O TIB: PHE 66: N, CA,
C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: ALA 68: N, CA, C, O, CB
TIB: ILE 76: N, CA, C, O, CB, CG1, CG2, CD1 TIB: VAL 77: N, CA, C,
O, CB, CG1, CG2 TIB: LEU 78: N, CA, C, O, CB, CG, CD1, CD2 TIB: SER
79: N, CA, C, O, CB, OG TIB: ASN 88: N, CA, C, O, CB, CG, OD1, ND2
TIB: GLY 91: N, CA, C, O TIB: ASN 92: N, CA, C, O, CB, CG, OD1, ND2
TIB: LEU 93: N, CA, C, O, CB, CG, CD1, CD2 TIB: ILE 100: N, CA, C,
O, CB, CG1, CG2, CD1 TIB: ASN 101: N, CA, C, O, CB, CG, OD1, ND2
TIB: ASP 102: N, CA, C, O, CB, CG, OD1, OD2 TIB: ILE 103: N, CA, C,
O, CB, CG1, CG2, CD1 TIB: CYS 104: N, CA, C, O, CB, SG TIB: SER
105: N, CA, C, O, CB, OG TIB: GLY 106: N, CA, C, O TIB: CYS 107: N,
CA, C, O, CB, SG TIB: SER 116: N, CA, C, O, CB, OG TIB: TRP 117: N,
CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2 TIB: SER
119: N, CA, C, O, CB, OG TIB: VAL 120: N, CA, C, O, CB, CG1, CG2
TIB: ALA 121: N, CA, C, O, CB TIB: VAL 132: N, CA, C, O, CB, CG1,
CG2 TIB: ARG 133: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 TIB:
GLU 134: N, CA, C, O, CB, CG, CD, OE1, OE2 TIB: PRO 136: N, CA, CD,
C, O, CB, CG TIB: ARG 139: N, CA, C, O, CB, CG, CD, NE, CZ, NH1,
NH2 TIB: VAL 140: N, CA, C, O, CB, CG1, CG2 TIB: VAL 141: N, CA, C,
O, CB, CG1, CG2 TIB: PHE 142: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ TIB: VAL 154: N, CA, C, O, CB, CG1, CG2 TIB: ALA 155: N,
CA, C, O, CB TIB: GLY 156: N, CA, C, O TIB: ALA 157: N, CA, C, O,
CB TIB: ASP 158: N, CA, C, O, CB, CG, OD1, OD2 TIB: LEU 159: N, CA,
C, O, CB, CG, CD1, CD2 TIB: ARG 160: N, CA, C, O, CB, CG, CD, NE,
CZ, NH1, NH2 TIB: GLY 161: N, CA, C, O TIB: ASN 162: N, CA, C, O,
CB, CG, OD1, ND2 TIB: GLY 163: N, CA, C, O TIB: TYR 164: N, CA, C,
O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH TIB: ASP 165: N, CA, C, O,
CB, CG, OD1, OD2 TIB: ILE 166: N, CA, C, O, CB, CG1, CG2, CD1 TIB:
ASP 167: N, CA, C, O, CB, CG, OD1, OD2 TIB: VAL 168: N, CA, C, O,
CB, CG1, CG2 TIB: PHE 169: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ TIB: GLY 177: N, CA, C, O TIB: ASN 178: N, CA, C, O, CB, CG,
CD1, ND2 TIB: ARG 179: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
TIB: ALA 180: N, CA, C, O, CB TIB: PHE 181: N, CA, C, O, CB, CG,
CD1, CD2, CE1, CE2, CZ TIB: ALA 182: N, CA, C, O, CB TIB: GLU 183:
N, CA, C, O, CB, CG, CD, OE1, OE2 TIB: PHE 184: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ TIB: LEU 185: N, CA, C, O, CB, CG, CD1,
CD2 TIB: VAL 187: N, CA, C, O, CB, CG1, CG2 TIB: THR 189: N, CA, C,
O, CB, OG1, CG2 TIB: GLY 190: N, CA, C, O TIB: GLY 191: N, CA, C, O
TIB: PRO 207: N, CA, CD, C, O, CB, CG TIB: PRO 208: N, CA, CD, C,
O, CB, CG TIB: ARG 209: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
TIB: GLU 210: N, CA, C, O, CB, CG, CD, OE1, OE2 TIB: PHE 211: N,
CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: GLY 212: N, CA, C, O
TIB: SER 214: N, CA, C, O, CB, OG TIB: HIS 215: N, CA, C, O, CB,
CG, ND1, CD2, CE1, ME2 TIB: SER 216: N, CA, C, O, CB, OG TIB: GLY
225: N, CA, C, O TIB: LEU 227: N, CA, C, O, CB, CG, CD1, CD2 TIB:
VAL 228: N, CA, C, O, CB, CG1, CG2 TIB: PRO 229: N, CA, CD, C, O,
CB, CG TIB: ILE 241: N, CA, C, O, CB, CG1, CG2, CD1 TIB: ASP 242:
N, CA, C, O, CB, CG, OD1, OD2 TIB: ALA 243: N, CA, C, O, CB TIB:
THR 244: N, CA, C, O, CB, OG1, CG2 TIB: PRO 250: N, CA, CD, C, O,
CB, CG TIB: PHE 262: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
TIB: CYS 268: N, CA, C, O, CB, SG Subset SUB5B: sub5mole.list
Subset SUB5B: TIB: 3-4, 6-7, 10-12, 15, 22-23, 25-30, 35, 37, 39,
41-42, 44-47, 51-55, 67, 69-70, TIB: 72, 74-75, 94-99, 108-112,
114-115, 118, 122-126, 128-131, 135, 137-138, TIB: 186, 188,
192-195, 213, 217-219, 223-224, 230-231, 234-235, 238-240, TIB:
245, 269 sub5batom.list Subset SUB5B: TIB: SER 3: N, CA, C, O, CB,
OG TIB: GLN 4: N, CA, C, O, CB, CG, CD, OE1, NE2 TIB: LEU 6: N, CA,
C, O, CB, CG, CD1, CD2 TIB: PHE 7: N, CA, C, O, CB, CG, CD1, CD2,
CE1, CE2, CZ TIB: PHE 10: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ TIB: ASN 11: N, CA, C, O, CB, CG, OD1, ND2 TIB: LEU 12: N, CA,
C, O, CB, CG, CD1, CD2 TIB: GLN 15: N, CA, C, O, CB, CG, CD, OE1,
NE2 TIB: CYS 22: N, CA, C, O, CB, SG TIB: GLY 23: N, CA, C, O TIB:
ASN 25: N, CA, C, O, CB, CG, OD1, ND2 TIB: ASN 26: N, CA, C, O, CB,
CG, OD1, ND2 TIB: ASP 27: N, CA, C, O, CB, CG, OD1, OD2 TIB: ALA
28: N, CA, C, O, CB TIB: PRO 29: N, CA, CD, C, O, CB, CG TIB: ALA
30: N, CA, C, O, CB TIB: THR 35: N, CA, C, O, CB, OG1, CG2 TIB: THR
37: N, CA, C, O, CB, OG1, CG2 TIB: ASN 39: N, CA, C, O, CB, CG,
OD1, ND2 TIB: CYS 41: N, CA, C, O, CB, SG TIB: PRO 42: N, CA, CD,
C, O, CB, CG TIB: VAL 44: N, CA, C, O, CB, CG1, CG2 TIB: GLU 45: N,
CA, C, O, CB, CG, CD, OE1, OE2 TIB: LYS 46: N, CA, C, O, CB, CG,
CD, CE, NZ TIB: ALA 47: N, CA, C, O, CB TIB: PHE 51: N, CA, C, O,
CB, CG, CD1, CD2, CE1, CE2, CZ TIB: LEU 52: N, CA, C, O, CB, CG,
CD1, CD2 TIB: TYR 53: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ,
OH TIB: SER 54: N, CA, C, O, CB, OG TIB: PHE 55: N, CA, C, O, CB,
CG, CD1, CD2, CE1, CE2, CZ TIB: LEU 67: N, CA, C, O, CB, CG, CD1,
CD2 TIB: LEU 69: N, CA, C, O, CB, CG, CD1, CD2 TIB: ASP 70: N, CA,
C, O, CB, CG, OD1, OD2 TIB: THR 72: N, CA, C, O, CB, OG1, CG2 TIB:
LYS 74: N, CA, C, O, CB, CG, CD, CE, NZ TIB: LEU 75: N, CA, C, O,
CB, CG, CD1, CD2 TIB: ASN 94: N, CA, C, O, CB, CG, OD1, ND2 TIB:
PHE 95: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: ASP 96: N,
CA, C, O, CB, CG, OD1, OD2 TIB: LEU 97: N, CA, C, O, CB, CG, CD1,
CD2 TIB: LYS 98: N, CA, C, O, CB, CG, CD, CE, NZ TIB: GLU 99: N,
CA, C, O, CB, CG, CD, OE1, OE2 TIB: ARG 108: N, CA, C, O, CB, CG,
CD, NE, CZ, NH1, NH2 TIB: GLY 109: N, CA, C, O TIB: HIS 110: N, CA,
C, O, CB, CG, ND1, CD2, CE1, NE2 TIB: ASP 111: N, CA, C, O, CB, CG,
OD1, OD2 TIB: GLY 112: N, CA, C, O TIB: THR 114: N, CA, C, O, CB,
OG1, CG2 TIB: SER 115: N, CA, C, O, CB, OG TIB: ARG 118: N, CA, C,
O, CB, CG, CD, NE, CZ, NH1, NH2 TIB: ASP 122: N, CA, C, O, CB, CG,
OD1, OD2 TIB: THR 123: N, CA, C, O, CB, OG1, CG2 TIB: LEU 124: N,
CA, C, O, CB, CG, CD1, CD2 TIB: ARG 125: N, CA, C, O, CB, CG, CD,
NE, CZ, NH1, NH2 TIB: GLN 126: N, CA, C, O, CB, CG, CD, OE1, NE2
TIB: VAL 128: N, CA, C, O, CB, CG1, CG2 TIB: GLU 129: N, CA, C, O,
CB, CG, CD, OE1, OE2 TIB: ASP 130: N, CA, C, O, CB, CG, OD1, OD2
TIB: ALA 131: N, CA, C, O, CB TIB: HIS 135: N, CA, C, O, CB, CG,
ND1, CD2, CE1, NE2 TIB: ASP 137: N, CA, C, O, CB, CG, OD1, OD2 TIB:
TYR 138: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH TIB: THR
186: N, CA, C, O, CB, OG1, CG2 TIB: GLN 188: N, CA, C, O, CB, CG,
CD, OE1, NE2 TIB: THR 192: N, CA, C, O, CB, OG1, CG2 TIB: LEU 193:
N, CA, C, O, CB, CG, CD1, CD2 TIB: TYR 194: N, CA, C, O, CB, CG,
CD1, CD2, CE1, CE2, CZ, OH TIB: ARG 195: N, CA, C, O, CB, CG, CD,
NE, CZ, NH1, NH2 TIB: TYR 213: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ, OH TIB: SER 217: N, CA, C, O, CB, OG TIB: PRO 218: N, CA,
CD, C, O, CB, CG TIB: GLU 219: N, CA, C, O, CB, CG, CD, OE1, OE2
TIB: LYS 223: N, CA, C, O, CB, CG, CD, CE, NZ TIB: SER 224: N, CA,
C, O, CB, OG TIB: VAL 230: N, CA, C, O, CB, CG1, CG2 TIB: THR 231:
N, CA, C, O, CB, OG1, CG2 TIB: ASP 234: N, CA, C, O, CB, CG, OD1,
OD2 TIB: ILE 235: N, CA, C, O, CB, CG1, CG2, CD1 TIB: ILE 238: N,
CA, C, O, CB, CG1, CG2, CD1 TIB: GLU 239: N, CA, C, O, CB, CG, CD,
OE1, OE2 TIB: GLY 240: N, CA, C, O TIB: GLY 245: N, CA, C, O TIB:
LEU 269: N, CA, C, O, CB, OXT, CG, CD1, CD2 Subset ACTSITE:
actsitemole.list Subset ACTSITE: TIB: 17, 21, 80-87, 89-90, 113,
143-153, 170-176, 196-206, 221-222, 226, 246-249, TIB: 251-261,
263-267 actsiteatom.list Subset ACTSITE: TIB: SER 17: N, CA, C, O,
CB, OG TIB: TYR 21: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
TIB: PHE 80: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: ARG
81: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 TIB: GLY 82: N, CA,
C, O TIB: SER 83: N, CA, C, O, CB, OG TIB: ARG 84: N, CA, C, O, CB,
CG, CD, NE, CZ, NH1, NH2 TIB: SER 85: N, CA, C, O, CB, OG TIB: ILE
86: N, CA, C, O, CB, CG1, CG2, CD1 TIB: GLU 87: N, CA, C, O, CB,
CG, CD, OE1, OE2 TIB: TRP 89: N, CA, C, O, CB, CG, CD1, CD2, NE1,
CE2, CE3, CZ2, CZ3, CH2 TIB: ILE 90: N, CA, C, O, CB, CG1, CG2, CD1
TIB: PHE 113: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ TIB: THR
143: N, CA, C, O, CB, OG1, CG2 TIB: GLY 144: N, CA, C, O TIB: HIS
145: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2 TIB: SER 146: N, CA,
C, O, CB, OG TIB: LEU 147: N, CA, C, O, CB, CG, CD1, CD2 TIB: GLY
148: N, CA, C, O TIB: GLY 149: N, CA, C, O TIB: ALA 150: N, CA, C,
O, CB TIB: LEU 151: N, CA, C, O, CB, CG, CD1, CD2 TIB: ALA 152: N,
CA, C, O, CB TIB: THR 153: N, CA, C, O, CB, OG1, CG2 TIB: SER 170:
N, CA, C, O, CB, OG TIB: TYR 171: N, CA, C, O, CB, CG, CD1,
CD2, CE1, CE2, CZ, OH TIB: GLY 172: N, CA, C, O TIB: ALA 173: N,
CA, C, O, CB TIB: PRO 174: N, CA, CD, C, O, CB, CG TIB: ARG 175: N,
CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 TIB: VAL 176: N, CA, C, O,
CB, CG1, CG2 TIB: ILE 196: N, CA, C, O, CB, CG1, CG2, CD1 TIB: THR
197: N, CA, C, O, CB, OG1, CG2 TIB: HIS 198: N, CA, C, O, CB, CG,
ND1, CD2, CE1, NE2 TIB: THR 199: N, CA, C, O, CB, OG1, CG2 TIB: ASN
200: N, CA, C, O, CB, CG, CD1, ND2 TIB: ASP 201: N, CA, C, O, CB,
CG, OD1, OD2 TIB: ILE 202: N, CA, C, O, CB, CG1, CG2, CD1 TIB: VAL
203: N, CA, C, O, CB, CG1, CG2 TIB: PRO 204: N, CA, CD, C, O, CB,
CG TIB: ARG 205: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2 TIB: LEU
206: N, CA, C, O, CB, CG, CD1, CD2 TIB: TRP 221: N, CA, C, O, CB,
CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2 TIB: ILE 222: N, CA, C,
O, CB, CG1, CG2, CD1 TIB: THR 226: N, CA, C, O, CB, OG1, CG2 TIB:
GLY 246: N, CA, C, O TIB: ASN 247: N, CA, C, O, CB, CG, OD1, ND2
TIB: ASN 248: N, CA, C, O, CB, CG, OD1, ND2 TIB: GLN 249: N, CA, C,
O, CB, CG, CD, OE1, NE2 TIB: ASN 251: N, CA, C, O, CB, CG, OD1, ND2
TIB: ILE 252: N, CA, C, O, CB, CG1, CG2, CD1 TIB: PRO 253: N, CA,
CD, C, O, CB, CG TIB: ASP 254: N, CA, C, O, CB, CG, OD1, OD2 TIB:
ILE 255: N, CA, C, O, CB, CG1, CG2, CD1 TIB: PRO 256: N, CA, CD, C,
O, CB, CG TIB: ALA 257: N, CA, C, O, CB TIB: HIS 258: N, CA, C, O,
CB, CG, ND1, CD2, CE1, NE2 TIB: LEU 259: N, CA, C, O, CB, CG, CD1,
CD2 TIB: TRP 260: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3,
CZ2, CZ3, CH2 TIB: TYR 261: N, CA, C, O, CB, CG, CD1, CD2, CE1,
CE2, CZ, OH TIB: GLY 263: N, CA, C, O TIB: LEU 264: N, CA, C, O,
CB, CG, CD1, CD2 TIB: ILE 265: N, CA, C, O, CB, CG1, CG2, CD1 TIB:
GLY 266: N, CA, C, O TIB: THR 267: N, CA, C, O, CB, OG1, CG2 Subset
RESTX: restxmole.list Subset RESTX: NEWMODEL: 14, 16, 18-20, 31-34,
36, 38, 40, 48-50, 56-66, 68, 78-79, 88, 91-93, NEWMODEL: 104-106,
120, 136, 225, 227-229, 250, 262, 268 restxatom.list Subset RESTX:
NEWMODEL: ALA 14: N, CA, C, O, CB NEWMODEL: TYR 16: N, CA, C, O,
CB, CG, CD1, CD2, CE1, CE2, CZ, OH NEWMODEL: ALA 18: N, CA, C, O,
CB NEWMODEL: ALA 19: N, CA, C, O, CB NEWMODEL: ALA 20: N, CA, C, O,
CB NEWMODEL: GLY 31: N, CA, C, O NEWMODEL: THR 32: N, CA, C, O, CB,
OG1, CG2 NEWMODEL: ASN 33: N, CA, C, O, CB, CG, OD1, ND2 NEWMODEL:
ILE 34: N, CA, C, O, CB, CG1, CG2, CD1 NEWMODEL: CYS 36: N, CA, C,
O, CB, SG NEWMODEL: GLY 38: N, CA, C, O NEWMODEL: ALA 40: N, CA, C,
O, CB NEWMODEL: ASP 48: N, CA, C, O, CB, CG, OD1, OD2 NEWMODEL: ALA
49: N, CA, C, O, CB NEWMODEL: THR 50: N, CA, C, O, CB, OG1, CG2
NEWMODEL: GLU 56: N, CA, C, O, CB, CG, CD, OE1, OE2 NEWMODEL: ASP
57: N, CA, C, O, CB, CG, OD1, OD2 NEWMODEL: SER 58: N, CA, C, O,
CB, OG NEWMODEL: GLY 59: N, CA, C, O NEWMODEL: VAL 60: N, CA, C, O,
CB, CG1, CG2 NEWMODEL: GLY 61: N, CA, C, O NEWMODEL: ASP 62: N, CA,
C, O, CB, CG, OD1, OD2 NEWMODEL: VAL 63: N, CA, C, O, CB, CG1, CG2
NEWMODEL: THR 64: N, CA, C, O, CB, OG1, CG2 NEWMODEL: GLY 65: N,
CA, C, O NEWMODEL: PHE 66: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2,
CZ NEWMODEL: ALA 68: N, CA, C, O, CB NEWMODEL: LEU 78: N, CA, C, O,
CB, CG, CD1, CD2 NEWMODEL: SER 79: N, CA, C, O, CB, OG NEWMODEL:
ASN 88: N, CA, C, O, CB, CG, OD1, ND2 NEWMODEL: GLY 91: N, CA, C, O
NEWMODEL: ASN 92: N, CA, C, O, CB, CG, OD1, ND2 NEWMODEL: LEU 93:
N, CA, C, O, CB, CG, CD1, CD2 NEWMODEL: CYS 104: N, CA, C, O, CB,
SG NEWMODEL: SER 105: N, CA, C, O, CB, OG NEWMODEL: GLY 106: N, CA,
C, O NEWMODEL: VAL 120: N, CA, C, O, CB, CG1, CG2 NEWMODEL: PRO
136: N, CA, CD, C, O, CB, CG NEWMODEL: GLY 225: N, CA, C, O
NEWMODEL: LEU 227: N, CA, C, O, CB, CG, CD1, CD2 NEWMODEL: VAL 228:
N, CA, C, O, CB, CG1, CG2 NEWMODEL: PRO 229: N, CA, CD, C, O, CB,
CG NEWMODEL: PRO 250: N, CA, CD, C, O, CB, CG NEWMODEL: PHE 262: N,
CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ NEWMODEL: CYS 268: N, CA,
C, O, CB, SG
Example 11
[0824] Providing a Lipase Variant E87K.sup.+ D254K
[0825] The Humicola lanuginosa lipase variant E87K.sup.+ D254K was
constructed, expressed and purified as described in WO
92/05249.
Example 12
[0826] Lipase-S-PEG 15,000 Conjugate
[0827] The lipase variant E87K.sup.+ D254K-SPEG conjugate was
prepared as described in Example 7, except that the enzyme is the
Humicola lanuginosa lipase variant (E87K.sup.+ D254K) described in
Example 11 and the polymer is mPEG 15,000.
Example 13
[0828] Immunogenicity Assessed as IgG.sub.1 of Lipase Variant
(D87K.sup.+ D254K) in Balb/C Mice
[0829] Balb/c mice were immunized by subcutaneous injection of:
[0830] (i) 50 microliters 0.9% (wt/vol) NaCl solution (control
group, 8 mice) (control),
[0831] (ii) 50 microliters 0.9% (wt/vol) NaCl solution containing
25 micrograms of protein of a Humicola lanuginosa lipase variant
(E87K.sup.+ D254K) (group 1, 8 mice) (unmodified lipase
variant),
[0832] (iii) 50% 0.9% (wt/vol) NaCl solution containing a Humicola
lanugoinosa lipase variant substituted in position D87K.sup.+ D254K
and coupled to an N-succinimidyl carbonate activated mPEG
15,000(group 2, 8 mice) (lipase-SPEG 15,000).
[0833] The amount of protein for each batch was measured by optical
density measurements. Blood samples (200 microliters) were
collected from the eyes one week after the immunization, but before
the following immunization. Serum was obtained by blood clotting,
and centrifugation.
[0834] The IgG.sub.1 response was determined by use of the Balb/C
mice IgG.sub.1 ELISA method as described above.
[0835] Results:
[0836] Five weekly immunizations were required to elicit a
detectable humoral response to the unmodified Humicola lanuginosa
variant. The antibody titers elicited by the conjugate (i.e.
lipase-SPEG15,000 ranged between 960 and 1920, and were only 2 to
4.times. lower than the antibody titer of 3840 that was elicited by
unmodified HL82-LIPOLASE (figure to the left).
[0837] The results of the tests are shown in FIG. 1.
[0838] As will be apparent to those skilled in the art, in the
light of the foregoing disclosure, many alterations and
modifications are possible in the practice of this invention
without departing from the spirit or scope thereof. Accordingly,
the scope of the invention is to be construed in accordance with
the substance defined by the following claims.
Sequence CWU 1
1
9 1 840 DNA bacillus sp. CDS (1)...(840) 1 tgg tca ccg aat gac cct
tac tat tct gct tac cag tat gga cca caa 48 Trp Ser Pro Asn Asp Pro
Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 1 5 10 15 aac acc tca acc
cct gct gcc tgg gat gta acc cgt gga agc agc act 96 Asn Thr Ser Thr
Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr 20 25 30 caa acg
gtg gcg gtc ctt gat tcc gga gtg gat tat aac cac cct gat 144 Gln Thr
Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp 35 40 45
ctt gca aga aaa gta ata aaa ggg tac gac ttt atc gac agg gac aat 192
Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn 50
55 60 aac cca atg gat ctt aac gga cat ggt acc cat gtt gcc ggt act
gtt 240 Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr
Val 65 70 75 80 gct gct gat acg aac aat gga att ggc gta gcc ggt atg
gca cca gat 288 Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met
Ala Pro Asp 85 90 95 acg aag atc ctt gcc gta cgg gtc ctt gat gcc
aat gga agt ggc tca 336 Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala
Asn Gly Ser Gly Ser 100 105 110 ctt gac agc att gcc tca ggt atc cgc
tat gct gct gat caa ggg gca 384 Leu Asp Ser Ile Ala Ser Gly Ile Arg
Tyr Ala Ala Asp Gln Gly Ala 115 120 125 aag gta ctc aac ctc tcc ctt
ggt tgc gaa tgc aac tcc aca act ctt 432 Lys Val Leu Asn Leu Ser Leu
Gly Cys Glu Cys Asn Ser Thr Thr Leu 130 135 140 aag agt gcc gtc gac
tat gca tgg aac aaa gga gct gta gtc gtt gct 480 Lys Ser Ala Val Asp
Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala 145 150 155 160 gct gca
ggg aat gac aat gta tcc cgt aca ttc caa cca gct tct tac 528 Ala Ala
Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr 165 170 175
cct aat gcc att gca gta ggt gcc att gac tcc aat gat cga aaa gca 576
Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala 180
185 190 tca ttc tcc aat tac gga acg tgg gtg gat gtc act gct cca ggt
gtg 624 Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly
Val 195 200 205 aac ata gca tca acc gtt ccg aat aat ggc tac tcc tac
atg tct ggt 672 Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr
Met Ser Gly 210 215 220 acg tcc atg gca tcc cct cac gtg gcc ggt ttg
gct gct ttg ttg gca 720 Thr Ser Met Ala Ser Pro His Val Ala Gly Leu
Ala Ala Leu Leu Ala 225 230 235 240 agt caa ggt aag aat aac gta caa
atc cgc cag gcc att gag caa acc 768 Ser Gln Gly Lys Asn Asn Val Gln
Ile Arg Gln Ala Ile Glu Gln Thr 245 250 255 gcc gat aag atc tct ggc
act gga aca aac ttc aag tat ggt aaa atc 816 Ala Asp Lys Ile Ser Gly
Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile 260 265 270 aac tca aac aaa
gct gta aga tac 840 Asn Ser Asn Lys Ala Val Arg Tyr 275 280 2 280
PRT bacillus sp. 2 Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln
Tyr Gly Pro Gln 1 5 10 15 Asn Thr Ser Thr Pro Ala Ala Trp Asp Val
Thr Arg Gly Ser Ser Thr 20 25 30 Gln Thr Val Ala Val Leu Asp Ser
Gly Val Asp Tyr Asn His Pro Asp 35 40 45 Leu Ala Arg Lys Val Ile
Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn 50 55 60 Asn Pro Met Asp
Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Ala Ala
Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp 85 90 95
Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser 100
105 110 Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly
Ala 115 120 125 Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser
Thr Thr Leu 130 135 140 Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly
Ala Val Val Val Ala 145 150 155 160 Ala Ala Gly Asn Asp Asn Val Ser
Arg Thr Phe Gln Pro Ala Ser Tyr 165 170 175 Pro Asn Ala Ile Ala Val
Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala 180 185 190 Ser Phe Ser Asn
Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val 195 200 205 Asn Ile
Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly 210 215 220
Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala 225
230 235 240 Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu
Gln Thr 245 250 255 Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys
Tyr Gly Lys Ile 260 265 270 Asn Ser Asn Lys Ala Val Arg Tyr 275 280
3 269 PRT Bacillus lentus 3 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg
Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser
Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His
Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly
Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val
Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85
90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp
Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly
Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser
Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn
Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala
Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg
Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala
Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210
215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln
Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly
Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala
Ala Thr Arg 260 265 4 344 PRT Arthromyces ramosus 4 Gln Gly Pro Gly
Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln 1 5 10 15 Ser Thr
Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp 20 25 30
Leu Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg 35
40 45 Lys Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro
Ala 50 55 60 Leu Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp
Gly Ser Ile 65 70 75 80 Ile Ala His Ser Asn Ile Glu Leu Ala Phe Pro
Ala Asn Gly Gly Leu 85 90 95 Thr Asp Thr Ile Glu Ala Leu Arg Ala
Val Gly Ile Asn His Gly Val 100 105 110 Ser Phe Gly Asp Leu Ile Gln
Phe Ala Thr Ala Val Gly Met Ser Asn 115 120 125 Cys Pro Gly Ser Pro
Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser 130 135 140 Ser Gln Pro
Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val 145 150 155 160
Thr Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu 165
170 175 Val Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly
Leu 180 185 190 Asn Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro
Gln Val Phe 195 200 205 Asp Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys
Gly Thr Thr Gln Pro 210 215 220 Gly Pro Ser Leu Gly Phe Ala Glu Glu
Leu Ser Pro Phe Pro Gly Glu 225 230 235 240 Phe Arg Met Arg Ser Asp
Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala 245 250 255 Cys Arg Trp Gln
Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg 260 265 270 Tyr Arg
Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn 275 280 285
Ala Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn 290
295 300 Ala Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu
Val 305 310 315 320 Ser Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr
Ala Ser Gly Pro 325 330 335 Leu Pro Ser Leu Ala Pro Ala Pro 340 5
876 DNA Humicola lanuginosa DSM 4109 CDS (1)...(876) sig_peptide
(1)...(66) mat_peptide (67)...(876) 5 atg agg agc tcc ctt gtg ctg
ttc ttt gtc tct gcg tgg acg gcc ttg 48 Met Arg Ser Ser Leu Val Leu
Phe Phe Val Ser Ala Trp Thr Ala Leu -20 -15 -10 gcc agt cct att cgt
cga gag gtc tcg cag gat ctg ttt aac cag ttc 96 Ala Ser Pro Ile Arg
Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe -5 1 5 10 aat ctc ttt
gca cag tat tct gca gcc gca tac tgc gga aaa aac aat 144 Asn Leu Phe
Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 15 20 25 gat
gcc cca gct ggt aca aac att acg tgc acg gga aat gcc tgc ccc 192 Asp
Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 30 35
40 gag gta gag aag gcg gat gca acg ttt ctc tac tcg ttt gaa gac tct
240 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55 gga gtg ggc gat gtc acc ggc ttc ctt gct ctc gac aac acg
aac aaa 288 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr
Asn Lys 60 65 70 ttg atc gtc ctc tct ttc cgt ggc tct cgt tcc ata
gag aac tgg atc 336 Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile
Glu Asn Trp Ile 75 80 85 90 ggg aat ctt aac ttc gac ttg aaa gaa ata
aat gac att tgc tcc ggc 384 Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile
Asn Asp Ile Cys Ser Gly 95 100 105 tgc agg gga cat gac ggc ttc act
tcg tcc tgg agg tct gta gcc gat 432 Cys Arg Gly His Asp Gly Phe Thr
Ser Ser Trp Arg Ser Val Ala Asp 110 115 120 acg tta agg cag aag gtg
gag gat gct gtg agg gag cat ccc gac tat 480 Thr Leu Arg Gln Lys Val
Glu Asp Ala Val Arg Glu His Pro Asp Tyr 125 130 135 cgc gtg gtg ttt
acc gga cat agc ttg ggt ggt gca ttg gca act gtt 528 Arg Val Val Phe
Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 140 145 150 gcc gga
gca gac ctg cgt gga aat ggg tat gat atc gac gtg ttt tca 576 Ala Gly
Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 155 160 165
170 tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc
624 Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185 gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat
gat att 672 Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn
Asp Ile 190 195 200 gtc cct aga ctc ccg ccg cgc gaa ttc ggt tac agc
cat tct agc cca 720 Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser
His Ser Ser Pro 205 210 215 gag tac tgg atc aaa tct gga acc ctt gtc
ccc gtc acc cga aac gat 768 Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val
Pro Val Thr Arg Asn Asp 220 225 230 atc gtg aag ata gaa ggc atc gat
gcc acc ggc ggc aat aac cag cct 816 Ile Val Lys Ile Glu Gly Ile Asp
Ala Thr Gly Gly Asn Asn Gln Pro 235 240 245 250 aac att ccg gat atc
cct gcg cac cta tgg tac ttc ggg tta att ggg 864 Asn Ile Pro Asp Ile
Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 255 260 265 aca tgt ctt
tag 876 Thr Cys Leu 6 291 PRT Humicola lanuginosa DSM 4109 SIGNAL
(1)...(22) 6 Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp
Thr Ala Leu -20 -15 -10 Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp
Leu Phe Asn Gln Phe -5 1 5 10 Asn Leu Phe Ala Gln Tyr Ser Ala Ala
Ala Tyr Cys Gly Lys Asn Asn 15 20 25 Asp Ala Pro Ala Gly Thr Asn
Ile Thr Cys Thr Gly Asn Ala Cys Pro 30 35 40 Glu Val Glu Lys Ala
Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 45 50 55 Gly Val Gly
Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys 60 65 70 Leu
Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile 75 80
85 90 Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser
Gly 95 100 105 Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser
Val Ala Asp 110 115 120 Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg
Glu His Pro Asp Tyr 125 130 135 Arg Val Val Phe Thr Gly His Ser Leu
Gly Gly Ala Leu Ala Thr Val 140 145 150 Ala Gly Ala Asp Leu Arg Gly
Asn Gly Tyr Asp Ile Asp Val Phe Ser 155 160 165 170 Tyr Gly Ala Pro
Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 175 180 185 Val Gln
Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 190 195 200
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 205
210 215 Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn
Asp 220 225 230 Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn
Asn Gln Pro 235 240 245 250 Asn Ile Pro Asp Ile Pro Ala His Leu Trp
Tyr Phe Gly Leu Ile Gly 255 260 265 Thr Cys Leu 7 32 DNA R28K oligo
7 gggatgtaac caagggaagc agcactcaaa cg 32 8 27 DNA R169K oligo 8
cgactttatc gataaggaca ataaccc 27 9 27 DNA R169K oligo 9 caatgtatcc
aaaacgttcc aaccagc 27
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