U.S. patent application number 11/751331 was filed with the patent office on 2010-03-18 for process for preparing an oil-in-water emulsion stabilised with recombinant collagen-like material and products prepared.
This patent application is currently assigned to FUJI FILM MANUFACTURING EUROPE B.V.. Invention is credited to Tanja Jacoba BOSCH VAN DEN, Jan Bastiaan BOUWSTRA, Joseph Hubertus OLIJVE, Yuzo TODA, Marc Willem Theodoor WERTEN, Richele Deodata WIND, Hendrik Wouter WISSELINK, Frederik Anton WOLF DE.
Application Number | 20100069512 11/751331 |
Document ID | / |
Family ID | 29286288 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100069512 |
Kind Code |
A1 |
OLIJVE; Joseph Hubertus ; et
al. |
March 18, 2010 |
PROCESS FOR PREPARING AN OIL-IN-WATER EMULSION STABILISED WITH
RECOMBINANT COLLAGEN-LIKE MATERIAL AND PRODUCTS PREPARED
Abstract
The invention provides oil-in-water emulsions comprising
recombinant collagen-like polymer in an amount sufficient to act as
stabiliser of the emulsion. The polymer is especially a polypeptide
which is free of helix structure, has an isoelectric point at least
0.5 pH units removed from the pH of the oil-in-water emulsion.
Furthermore, bipolar recombinant collagen-like polymers are
provided for use in oil-in-water emulsions. The bipolar polymers
are polar at one end as a result of a relative abundance of polar
amino acids, and apolar at the other end as a result of a relative
abundance of apolar amino acids.
Inventors: |
OLIJVE; Joseph Hubertus;
(KAATSHEUVEL, NL) ; BOUWSTRA; Jan Bastiaan;
(BILTHOVEN, NL) ; WOLF DE; Frederik Anton;
(BUNNIK, NL) ; WERTEN; Marc Willem Theodoor;
(WAGENINGEN, NL) ; WISSELINK; Hendrik Wouter;
(NIJMEGEN, NL) ; WIND; Richele Deodata; (WIJCHEN,
NL) ; BOSCH VAN DEN; Tanja Jacoba; (EDE, NL) ;
TODA; Yuzo; (GOIRLE, NL) |
Correspondence
Address: |
IP Patent Docketing;K&L GATES LLP
599 Lexington Avenue, 33rd Floor
New York
NY
10022-6030
US
|
Assignee: |
FUJI FILM MANUFACTURING EUROPE
B.V.
TILBURG
NL
|
Family ID: |
29286288 |
Appl. No.: |
11/751331 |
Filed: |
May 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10672040 |
Sep 26, 2003 |
7229631 |
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11751331 |
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09602459 |
Jun 23, 2000 |
6645712 |
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10672040 |
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Current U.S.
Class: |
514/774 ;
252/182.12 |
Current CPC
Class: |
G03C 1/005 20130101 |
Class at
Publication: |
514/774 ;
252/182.12 |
International
Class: |
A61K 47/42 20060101
A61K047/42; C09K 3/00 20060101 C09K003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 1999 |
EP |
99202047.9 |
Claims
1-22. (canceled)
23. A process for preparing an oil-in-water-emulsion comprising
mixing a hydrophilic phase and a hydrophobic phase, wherein the
hydrophilic phase comprises a collagen-like recombinant peptide in
an amount sufficient to stabilize the emulsion, the collagen-like
recombinant peptide comprising at least one GXY domain having a
length of at least 5 consecutive GXY triplets, wherein each X and
each Y represents an amino acid residue, and wherein at least 20
percent of the amino acid residues of the recombinant collagen-like
peptide are constituents of consecutive GXY triplets, and the
hydrophobic phase comprises an additive selected from the group
consisting of a pharmaceutical agent, a nutritional agent and a
photographic agent.
24. Process according to claim 23, wherein the recombinant
collagen-like peptide is free of helix structure.
25. Process according to claim 23, wherein the recombinant
collagen-like polymer is free of hydroxyproline residues.
26. Process according to claim 23, wherein the recombinant
collagen-like peptide has an isoelectric point at least 0.5 pH
units removed from the pH of the oil-in-water emulsion.
27. Process according to claim 23, wherein the recombinant
collagen-like peptide has an isoelectric point of 4 or 10 or in the
range between 4 and 10.
28. Process according to claim 23, wherein the recombinant
collagen-like peptide has a molecular weight in the range of from
2.5 kDa to 100 kDa.
29. Process according to claim 23, wherein the recombinant
collagen-like peptide is homodisperse with regard to the molecular
weight of the peptide.
30. Process according to claim 23, wherein the hydrophilic phase
further comprises non-recombinant collagen.
31. Process according to claim 23, wherein the recombinant
collagen-like peptide exhibits an amphiphilic structure and
comprises at least one polar part containing at least 10 polar
amino acid residues and at least one apolar part containing at
least 10 apolar amino acid residues.
32. Process according to claim 31, wherein the lengths of the at
least one polar part and of the at least one apolar part are each
at least 10 percent of the length of the peptide backbone.
33. Process according to claim 31, wherein the average transfer
free energy per amino acid residue of the at least one polar part
is at least 0.3 kcal/mole lower than the average transfer free
energy per amino acid of the at least one apolar part.
34. Process according to claim 23, wherein the oil-in-water
emulsion exhibits an initial droplet size of less than 500 nm at a
temperature of 40.degree. C. or lower and at a pH of 5.
35. Process according to claim 33, wherein the oil-in-water
emulsion exhibits an increase in droplet size of less than 400 nm
after 4 hours at a temperature of 40.degree. C. or lower and at a
pH of 5.
36. Process according to claim 23, wherein the recombinant
collagen-like peptide is present in a solvent in a concentration in
the range from about 2 g/l to about 100 g/l of solvent.
37. Process according to claim 23, wherein the recombinant
collagen-like peptide exhibits a viscosity in the range of from
0.005 mP to 8 mP when dissolved at a concentration of 6.6 percent
in water at a temperature of 40.degree. C.
38. Process according to claim 23, wherein the recombinant
collagen-like peptide does not exhibit gelation at a temperature
below 30.degree. C.
39. Process according to claim 23, wherein the hydrophobic phase
additive is selected from the group consisting of trihexyl-,
trioctyl-, tridecyl-, tris(butoxyethyl)-, tris(haloalkyl)-,
trixylenyl- and tricresyl-phosphates.
40. Process according to claim 23, wherein the hydrophobic phase
additive comprises edible triglycerides derived from vegetable or
animal fats.
41. Process according to claim 23, wherein the hydrophobic phase
additive comprises sodium dodecylbenzenesulphonate.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 10/672,040, filed Sep. 26, 2003, which is a divisional of U.S.
application Ser. No. 09/602,459, filed Jun. 23, 2000, now U.S. Pat.
No. 6,645,712, the entire disclosure of which application is hereby
incorporated herein by reference thereto.
SUMMARY OF THE INVENTION
[0002] The subject invention is directed at an oil-in-water
emulsion in which recombinant collagen-like polymer is applied as
stabiliser. The stabilising effect occurs already at the stage of
formation i.e. on the initial size of the droplets in the emulsion.
Also the stabilising effect is visible when assessing the ageing of
the oil-in-water emulsion. In both cases the droplet size is
significantly reduced vis-a-vis the prior art oil-in-water
emulsions comprising gelatin. The stabilising effect occurs at a
range of temperatures and a range of pH values. It now in fact has
become possible to operate processes requiring oil-in-water
emulsions at lower temperatures than was possible to date and also
at lower pH values than normally are applied to date. The same
holds true for the storage temperature and pH at which the
oil-in-water emulsions according to the invention can currently be
maintained. The oil-in-water emulsions can now be stored longer
than was previously the case. Also the oil-in-water emulsions
according to the invention can be as stable as the prior art
oil-in-water emulsions comprising gelatin at lower concentrations
of surfactant than used in the prior art oil-in-water
emulsions.
[0003] In addition the subject invention provides the possibility
to use recombinant collagen-like polymer which are composed of
polar and apolar end tails for oil-in-water emulsions and also
provides for the first time the bipolar, or more specifically
amphiphilic, compounds as such and a description of a method to
achieve the production of such compounds.
[0004] Oil-in-water emulsions consist of hydrophobic droplets in a
hydrophilic continuous phase. The interfacial area between these
hydrophobic droplets and the hydrophilic continuous phase is
stabilised with surfactants and/or polymers.
[0005] In the manufacturing process, the size of the droplets in
the oil-in-water emulsion is a factor needing careful control. The
average size of the droplets in the oil-in-water emulsion should be
small i.e. the initial size of the droplets should be as low as
possible. Also the size stability of the droplets after making the
oil-in-water emulsion should be high i.e. the ageing stability
should be as high as possible thus ensuring the increase in droplet
size in time is kept as low as possible. To realise this small
initial size and this limited ageing, the oil droplets can be
stabilised by gelatin.
[0006] Present stabilisation methods however have several
disadvantages:
1. The initial size of the oil-in-water emulsion is rather large.
2. The stabilisation capability of the present gelatin is limited,
meaning that in the manufacturing process the oil-in-water
emulsions have a limited life time in which they can be applied for
specific functions.
[0007] The presently used polymer-like materials (like gelatin)
originate from natural sources and the structure and the related
rheological and surface chemical characteristics can be modified
only in a limited manner. J. Colloid Polym. Sci. 272: 433-439 for
example reveals experimental data about the relation between the
molecular mass distribution of non-recombinant natural gelatin and
it's effectiveness in the stabilisation of oil-in-water emulsions.
In the case of gelatin samples with a content of more than 30% of
the low molecular weight fraction as described in the article an
improved stabilisation was obtained in comparison to the native non
recombinant non hydrolysed gelatin. The problem still remained with
this modified gelatin that the reproducibility of such processes
using natural gelatin sources is not extremely good. In particular
this is a preferred requirement for photographic applications.
[0008] In addition when considering use of oil-in-water emulsions
for consumption purposes e.g. in foodstuffs the risk associated
with mad cows disease for example can have a prohibitive effect on
the use of gelatin derived from natural sources as a
stabiliser.
BRIEF DESCRIPTION OF THE DRAWING
[0009] Some results obtainable with an embodiment of emulsion
employing a recombinant gelatin according to the invention,
compared with a conventional gelatin, are illustrated in the single
FIGURE of the accompanying drawing.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The invention is directed at an oil-in-water emulsion
comprising recombinant collagen-like (or gelatin-like) polymer in
an amount sufficient to act as stabiliser of the emulsion. The
advantages thereof are described in detail elsewhere in the
description. An oil-in-water emulsion according to the invention
suitably is one wherein the recombinant collagen-like polymer is
free of triple helix structure. The recombinant collagen-like
polymer is suitably free of any helix structure. It is a preferred
embodiment of the invention that the recombinant collagen-like
polymer of the oil-in-water emulsion is free of hydroxyproline as
this ensures the absence of (triple) helix formation. The triple
helical structure is present in natural gelatin. The absence of the
(triple) helical structure is advantageous, because the
emulsification can be operated at lower temperatures (15-40.degree.
C.) than the traditional temperature during the emulsification
process (T higher than 40.degree. C.).
[0011] The method of arriving at recombinant collagen-like polymer
has been described in detail in commonly owned U.S. Pat. No.
6,150,081, inventors van Heerde et al., for example at column 14,
line 48 to column 15, line 17, at column 22, line 51 to column 25
line 18 and elsewhere throughout the specification, the entire
disclosure of which patent is hereby incorporated herein by
reference thereto. The methodology is described in the publication
`High yield secretion of recombinant gelatins by Pichia pastoris`,
M. W. T. Werten et al., Yeast, 15, 1087-1096 (1999), in press.
[0012] To be defined as collagen-like at least one GXY domain
should be present of at least a length of 5 consecutive GXY
triplets and at least 20% of the amino acids of the recombinant
collagen-like polymer should be present in the form of consecutive
GXY triplets, wherein a GXY triplet consists of G representing
glycine and X and Y representing any amino acid. Suitably at least
5% of X and/or Y can represent proline and in particular at least
5%, more in particular between 10 and 33% of the amino acids of the
GXY part of the recombinant collagen-like polymer is proline. For
the purposes of this patent application the recombinant
collagen-like polymer consists of at least 4 different amino acids,
preferably more than 10 different amino acids, more preferably more
than 15 different amino acids. It can comprise any of the amino
acids known. A preferred oil-in-water emulsion according to the
invention is one, wherein the recombinant collagen-like polymer
comprises at least one lysine residue.
[0013] Any of the embodiments disclosed in the van Heerde et al.
U.S. Pat. No. 6,150,081 can be applied for the oil-in-water
emulsions according to the invention. A preferred embodiment of an
oil-in-water emulsion according to the invention is one wherein the
recombinant collagen-like polymer has an isoelectric point at least
0.5 pH units removed from the pH of the oil-in-water emulsion
itself, suitably one pH unit removed or even more. The advantage
hereof is that the pH at which the emulsion needs to be maintained
or used or prepared can vary depending on the isoelectric point
(pI) of the applied recombinant collagen-like polymer. The
recombinant technology enables variation previously unavailable for
tailoring the polymer and thus tailoring the pI. It will be
appreciated that not all processes requiring an oil-in-water
emulsion are best carried out at pH 6 which is the pH value at
which prior art gelatin comprising oil-in-water emulsions were
optimally used. Naturally the pH=6 can also be used in those cases
where it is still useful or in fact optimal. However the
oil-in-water emulsions according to the invention no longer need
the strict control of the pH during any of the processes e.g.
preparation, storage or application as was previously the case. Now
it has in addition become possible to use the oil-in-water
emulsions according to the invention at pH=5. It has now become
possible to develop oil-in-water emulsions with recombinant
collagen-like polymers of extremely divergent pI values. Suitable
embodiments involve pI anywhere from 4-10. pI equal to or higher
than 6, equal to or higher than 7 and even equal to or higher than
8 and higher than 9 have been achieved and they are illustrated in
the examples. We also illustrate pI selected from 4-7. The presence
of collagen-like polymers with an isoelectric point far from the
actual pH of the OW emulsion according to the invention is
preferred. Such a pH has the advantage that the overall charge and
the overall three dimensional conformation of the polymer is
independent of the pH, and so the steric stabilisation of the OW
emulsion is also independent of the pH.
[0014] An oil-in-water emulsion according to the invention will use
recombinant collagen-like polymer with a molecular weight of at
least 2.5 kDa. Suitably the molecular weight is lower than 170 kDa,
preferably lower than 100 kDa. We have found improved results when
the molecular weight is higher than 20 kDa, preferably higher than
25 kDa and even more preferably higher than 50 kDa. A preferred
range thus goes from 20 kDa to 100 kDa.
[0015] An oil-in-water emulsion according to the invention, which
is particularly useful, is one, wherein the recombinant
collagen-like polymer is present in a homodisperse size
distribution. A homodisperse size distribution means that the
optimal size distribution and the uniformity and reproducibility
can be guaranteed for the desired application. According to the
invention, the notion "homodisperse" preferably means that at least
75% of the molecules have a molecular weight between -10% and +10%
of the average molecular weight. It is clear for example that
steric hindrance is limited in cases where the size is too low. A
size that is too high causes a high viscosity, which is
inconvenient for the emulsion equipment and for the emulsification
process. The invention now provides the opportunity to regulate
this in an optimal manner. A lower viscosity enables application of
higher concentrations of the gelatin and the oil. Thus the ratio of
oil versus water can be improved, which will be advantageous for
several photographic applications.
[0016] In an alternative embodiment an oil-in-water emulsion
according to the invention, is one wherein the recombinant
collagen-like polymer is present together with non recombinant
collagen i.e. an oil-in-water emulsion which comprises a mix with
natural gelatin or prior art gelatins can also be used.
Surprisingly good results concerning stability vis-a-vis initial
size and ageing stability are possible. No phase separation occurs
and dissolving occurs only in the water, which is particularly
interesting for example in photographic application. In a suitable
embodiment the oil-in-water emulsion according to the invention can
be one, wherein the recombinant collagen-like polymer is present
together with non recombinant collagen in a ratio of 99%-20% on
weight basis of recombinant collagen-like polymer on the basis of
total weight of collagen-like polymer in the oil-in-water emulsion.
The initial size of the oil-in-water emulsion resulting from this
mixing process, stabilised by said protein-like material made by
genetic engineering, was smaller than the initial size of the
oil-in-water stabilised by traditional polymer-like material, and
the ageing characteristics of said oil-in-water emulsion were
improved, under a wide variety of conditions (variation in T,
surfactant, pH, polymer-like stabiliser combinations, etc.), as
compared with the prior art.
[0017] Of particular interest is the fact that oil-in-water
emulsions according to any of the embodiments of the invention
exhibit better initial size characteristics as can be determined by
measuring the droplet size at a particular pH and temperature of
the emulsion and measuring the size under the same conditions for a
prior art oil-in-water emulsion. A suitable test revealing better
initial size characteristics can comprise measuring a smaller
initial droplet size at T=40.degree. C. or less and pH=5 at 2 ml
scale using ultrasonic technique in comparison to prior art gelatin
under corresponding conditions, e.g. at a temperature T selected
from the range of 10-40.degree. C., suitably 15-40.degree. C. e.g.
T=30, 25, 20, 15 or 10.degree. C., wherein the comparison is
optionally carried out in the presence of surfactant, e.g. in an
amount corresponding to 0.4868 mM SDBS/5 grams of collagen-like
polymer/litre. An improvement can comprise the oil-in-water
emulsion according to the invention exhibiting better initial size
characteristics as can be determined by measuring a smaller initial
droplet size than 600 nm, preferably below 500 nm, even lower than
350 nm, 250 nm and more preferably below 200 nm at T=40.degree. C.
or less e.g. at a T selected from the range of 10-40, suitably
15-40.degree. C. e.g. T=30, 25, 20, 15 or 10.degree. C. at pH=5,
wherein the comparison is carried out optionally in the presence of
a surfactant, e.g. in an amount corresponding to 0.4868 mM SDBS/5
grams of collagen-like polymer per litre.
[0018] Not only is an improvement of initial size often found, but
also better ageing characteristics as can be determined by
measuring the droplet size after a period of time at a particular
pH and temperature of the emulsion and measuring the size under the
same conditions for a prior art oil-in-water emulsion. An example
of a suitable test to reveal this characteristic is by measuring an
increase in droplet size after 4 hours at T=40.degree. C. or less
and pH=6 at 2 ml scale using ultrasonic technique in comparison to
prior art gelatin under corresponding conditions, e.g. a T from the
range 10-40, suitably 15-40.degree. C. e.g. T=30, 25, 20, 15 or
10.degree. C., wherein the comparison is optionally carried out in
the presence of surfactant, e.g. in an amount corresponding to
0.4868 mM SDBS/5 grams of collagen-like polymer/litre. Suitably one
will find for oil-in-water emulsions according to the invention
better ageing characteristics as can be determined by measuring a
smaller increase in droplet size after 4 hours than 450 nm,
preferably below 400 nm, preferably below 350 nm and more
preferably below 300 nm and even below 250 nm at T=40.degree. C. or
less e.g. at a T selected from the range of 10-40.degree. C.,
suitably 15-40.degree. C. e.g. T=30, 25, 20, 15 or 10.degree. C. at
pH=6, wherein the comparison is carried out optionally in the
presence of surfactant, e.g. in an amount corresponding to 0.4868
mM SDBS/5 grams of collagen like polymer/litre.
[0019] The tests can be carried out at different pH values
depending on the recombinant collagen-like polymer used in the
oil-in-water emulsion. The improvement is generally more noticeable
at lower temperatures and at pH lower than those generally used for
prior art gelatins i.e. at a pH lower than 6 suitably lower than
5.5 e.g. around 5 or lower.
[0020] An oil-in-water emulsion according to any of the embodiments
of the invention will not exhibit gelation at a temperature below
30.degree. C.
[0021] Oil-in-water emulsions according to any of the embodiments
of the invention will exhibit increased stability in the presence
of surfactant at a concentration below that equivalent to 1 mmol
SDBS/5 gram gelatin/l as can be determined by measurement of
droplet size increase after 4 hours at pH 6.0 and T=40.degree. C.
below 250 nm.
[0022] An oil-in-water emulsion according to any of the embodiments
of the invention can comprise the recombinant collagen-like polymer
in concentrations of collagen-like polymer in the range of 2-100
gram/1 solvent in particular between 5 and 50 g/l solvent. This is
advantageous in comparison to the prior art oil-in-water emulsions
i.e. higher gelatin concentrations are feasible than oil-in-water
emulsions using gelatin applied in the prior art.
[0023] An oil-in-water emulsion according to any of the embodiments
of the invention can exhibit a viscosity in the range 0.005-8 mPa
when dissolved in a concentration of 6.6% in water at a temperature
of 40.degree. C.
[0024] Due to the development of the recombinant technology it has
now become possible to develop for use specifically in oil-in-water
emulsion according to any of the embodiments of the emulsions
according to the invention recombinant collagen-like polymer
exhibiting an amphiphilic structure, with one end of the molecule
being polar and the other end being apolar e.g. wherein the
recombinant collagen-like polymer exhibits an amphiphilic
structure, with one end of the molecule being polar due to the
presence of a sufficient number of polar amino acid residues to
render that end polar and the other end being apolar due to the
presence of a sufficient number of apolar amino acid residues to
render that end apolar. Collagen-like polymers with an amphipolar
character (one side hydrophilic, one side hydrophobic) show an
optimal interfacial behaviour and have a strong preference for a
position on the oil-water interface (with one leg in the oil-phase
and "one leg" in the water-phase, resulting in a low interfacial
tension) by which the initial size and stabilisation are optimised.
The manufacture of the polar hydrophilic collagen molecule can be
made following the detailed method described in van Heerde et al.
U.S. Pat. No. 6,150,081. Obviously the changes required in the
amino acid sequence can be achieved in a manner well known to the
skilled person when wishing to introduce a few specific amino acid
substitutions. The skilled person also knows which amino acids can
be substituted and which amino acids can be used to enhance
polarity or apolarity. The polar and apolar constructs can be
combined using standard methodologies of ligation for the
manufacture of the bi-functional collagen-like polymer. Not only is
an oil-in-water emulsion as such part of the invention but also any
of the bipolar molecules as such and a process for making them. An
amphiphilic recombinant collagen-like polymer i.e. polar at one end
and apolar at the other to a degree sufficient for the polar end to
extend into a water phase and the apolar end to extend into an oil
phase, wherein recombinant collagen-like is further as described
for any of the recombinant collagen-like polymers as components of
an oil-in-water emulsion according to the invention is thus also
covered.
[0025] The amphiphilic nature of the preferred collagen-like
polymers of the invention can be defined with reference to the
transfer free energy of the individual amino acids constituting the
polar and apolar parts of the polymer, respectively. This transfer
free energy (AF) is the energy (in kcal/mole) of the amino acid
residue in an .alpha.-helix to be transferred from the membrane
interior to the water phase. These energy values as defined by
Engelman et al, Ann. Rev. Biophys. Biophys. Chem. 15 (1986), 330,
are summarised in the table below.
TABLE-US-00001 a.a Phe Met Ile Leu Val Cys Trp Ala Thr Gly .DELTA.F
3.7 3.4 3.1 2.8 2.6 2 1.9 1.6 1.2 1.0 a.a. Ser Pro Tyr His Gln Asn
Glu Lys Asp Arg .DELTA.F 0.6 -0.2 -0.7 -3 -4.1 -4.8 -8.2 -8.8 -9.2
-12.3
[0026] The polarity of a given amino acid sequence is defined
herein as the average transfer free energy per amino acid of the
sequence, which equals the sum of the product of the number of
individual amino acids and the transfer free energy of each amino
acid, divided by the total number of amino acids. In a formula:
Polarity=(.SIGMA..sub.n.sub.i*.DELTA.F.sub.i)/n.sub.t
wherein n.sub.t is the number of each individual amino acid,
.DELTA.F.sub.i is the transfer free energy of the corresponding
amino acid, and n.sub.t is the total number of amino acids. As an
example, a 15-mer apolar peptide having the following amino acid
sequence:
[0027] Gly Pro Pro Gly Val Pro Gly Phe Ile Gly Phe Pro Gly Leu
Pro
has the following amino acid composition: 5 Gly+5 Pro+1 Val+2 Phe+1
Ile+1 Leu, and hence it has the following polarity (in kcal/mole
per amino acid):
(5*1.0+5*-0.2+1*2.6+2*3.7+1*3.1+1*2.8)/15=19.9/15=+1.33
[0028] Apolar sequences generally have positive polarity values,
whereas polar sequences have negative polarity values. According to
the invention, amphiphilic collagen-like polymers have a polar part
and an apolar part, the polar part having a polarity value which is
at least 0.3 lower (i.e. less positive or more negative),
preferably at least 0.5 lower, more preferably at least 0.7 lower
than the apolar part. The polar aprt an the apolar part may be
separated by a bridge, the polarity of which may be intermediate.
it is preferred that the polar part and apolar part each make up at
least 15% of the total length (defined in chain atom numbers) of
the polymer, preferably each at least 30% of the length. In
particular each part (polar and apolar) contains at least 15, more
in particular at least 30, most particularly at least 50 amino
acids, up to half of the total number of amino acids. Preferably
the polar and apolar parts are located at the two opposite ends of
the polymer, with preferably less than 5%, or less than 10 amino
acids, and most desirably no amino acids being located at the outer
ends beyond the polar and apolar parts.
[0029] In a preferred embodiment, the polar part of the amphiphilic
polymer contains at least 10% (on the basis of the number of amino
acids), preferably at least 15%, of polar amino acids selected from
Arg, Asp, Lys, Glu, Asn, Gln and His, whereas the apolar part
contains at least 10%, preferably at least 15%, of apolar amino
acids selected from Phe, Met, Ile, Leu, Val, Trp and Ala. In both
parts, at least about 15, preferably at least 30% will be Gly, and
at least 10% will be Pro. Preferably, the polar part contains less
than 10% (more preferably less than 7%) of the apolar amino acids
selected from Phe, Met, Ile, Leu, Val, Trp and Ala and the apolar
part contains less than 10% (more preferably less than 7%) of the
polar amino acids selected from Arg, Asp, Lys, Glu, Asn, Gln and
His.
[0030] The amphiphilic polymer may also comprise alternating polar
and apolar stretches, each stretch being e.g. between 5 and 100,
preferably between 10 and 50 amino acids in length. The number of
alternating stretches may be two up to e.g. ten of such stretches
(pairs of polar and apolar stretches). At least one polar stretch
of such series, preferably two or more stretches, more preferably
at least the terminal polar stretch, and most preferably each polar
stretch has a polarity difference with the apolar stretch or
preferably the apolar stretches as defined above. In this
alternating arrangement, each pair of polar and apolar stretches
may be separated from the next pair by an indifferent bridge of
intermediate polarity.
[0031] Obviously an oil-in-water emulsion according to any of the
embodiments of the invention described above as such or any
combination thereof is covered by the invention. Also any such
oil-in-water emulsion can comprise further additives rendering it
particularly suited to the application purpose of the emulsion. By
way of example for the preferred application in photography, said
additive can be selected from any of the following group of
components, said group consisting of coupler, dye, organic solvent,
inorganic solvent, surface/interface active agent, scavenger, UV
absorber, optical brightener, stabiliser, pH controlling agent,
mono/divalent ions. In the case of application in foodstuffs,
pharmaceuticals or cosmetics the additives must be non toxic i.e.
pharmacologically acceptable to humans and/or animals.
[0032] Examples of protein-like structures, which can be applied
for stabilisation of OW emulsions, are provided in the experimental
description elsewhere. In the examples the improvement of the
oil-in-water emulsion stability and the oil-in-water emulsion
initial size is illustrated by use of various recombinant
collagen-like polymers under various conditions. For example
homodisperse molecules of varying sizes have been used.
[0033] Molecules in which helical structure is absent have been
used. Molecules with an pI of 9 have been used. The pH dependence
and the T dependence of the OW emulsion stability and initial size
are shown.
[0034] Oil-in-water emulsions are made by mixing a solution of
collagen-like material in the hydrophilic phase with a hydrophobic
phase. Mixing can be executed by stirring, by high-pressure
homogenisation, by treatment with ultrasonic frequencies, or the
like. The hydrophobic phase can be any hydrophobic liquid suitable
for the intended use. For example, trialkyl phosphates and triaryl
phosphates such as trihexyl, trioctyl, tridecyl, tris(butoxyethyl),
tris(haloalkyl), trixylenyl and tricresyl phosphate, can be used
for preparing photographic emulsions. Also phthalate esters, citric
esters, benzoic esters, fatty acid esters and fatty acid amides, as
well as hydrocarbons such as n-decane or n-dodecane can be used.
Edible triglycerides derived from vegetable or animal fats can be
used for preparing emulsions for use in nutritional, cosmetic and
pharmaceutical products, etc. Surfactants, such as sodium
dodecylbenzenesulphonate, can be added and oil-soluble components
such as precursor molecules for dyes and UV absorbers, and further
reducing reducing agents and other compounds can be added.
Temperature can be varied. The protein-like material can consist of
a pure component (homodisperse) or a mixture of components, all
made by genetic engineering, or it can consist of a mixture of a
component made genetic engineering and a traditional polymer. The
invention covers a process comprising application of an
oil-in-water emulsion according to any of the embodiments provided
as oil-in-water emulsions according to the invention. Specifically
the process can be a photography process or a foodstuff production
process. Suitably a process according to the invention can be
carried out at least at some stage in the presence of the
oil-in-water emulsion at a pH below 6.0 preferably below 5.5 and
suitably between 4.5-5.5. A process according to the invention can
be carried out at some stage in the presence of the oil-in-water
emulsion at a temperature below 40.degree. C., suitably at ambient
temperature i.e. between 10-30.degree. C., suitably between
18-25.degree. C. i.e. in absence of a heating step, preferably
during the whole process. A process comprising a combination of any
of the steps mentioned falls within process protection claimed. A
process comprising any of the above mentioned measures, said
process being storage of an oil-in-water emulsion according to any
of the embodiments of the invention is also covered by the
invention as is a process of preparation of any of the embodiments
of the oil-in-water emulsion according to the invention.
General Remarks about Advantages of the Application of the
Recombinant Collagen-Like Polymers Over Traditional Gelatins:
[0035] Mono disperse products, creating the flexibility to design
an OW emulsion with an optimal MW mix for creating steric hindrance
without `bridge making" coagulation behaviour.
[0036] Prevention of gelation behaviour (when indicated), creating
freedom of processing temperature.
[0037] Freedom to choose the isoelectric point and surface active
behaviour (by polar/a-polar AA), which is for stabilisation and for
robustness of stability in case of emulsion pH variations
(=amphipolar collagen-like polymers).
[0038] Freedom to use lower surfactant concentrations to obtain
comparable or even improved stability.
[0039] The recombinant collagen like polypeptide as defined above
can be produced by expression of a collagen-like polypeptide
encoding nucleic acid sequence by a suitable microorganism. The
process can suitably be carried out with a fungal cell or a yeast
cell. Suitably the host cell is selected from the group consisting
of high expression host cells like Hansenula, Trichoderma,
Aspergillus, Penicillium, Neurospora and Pichia. Fungal and yeast
cells are preferred to bacteria as they are less susceptible to
improper expression of repetitive sequences. Most preferably the
host will not have a high level of proteases that attack the
collagen structure expressed. In this respect Pichia offers an
example of a very suitable expression system. Preferably the
micro-organism is free of active post-translational processing
mechanism for processing collagen like sequences to fibrils thereby
ensuring absence of helix structure in the expression product. Also
such a process can occur when the micro-organism is free of active
post-translational processing mechanism for processing collagen
like sequences to triple helices and/or when the nucleic acid
sequence to be expressed is free of procollagen and telopeptide
encoding sequences. The host to be used does not require the
presence of a gene for expression of prolyl-4-hydroxylase the
enzyme required for collagen triple helix assembly contrary to
previous suggestions in the art concerning collagen production. The
selection of a suitable host cell from known industrial enzyme
producing fungal host cells specifically yeast cells on the basis
of the required parameters described herein rendering the host cell
suitable for expression of recombinant collagen according to the
invention suitable for photographic applications in combination
with knowledge regarding the host cells and the sequence to be
expressed will be possible by a person skilled in the art.
[0040] Several strong and tightly-regulated inducible promoters are
available for yeast systems and other recombinant production
systems, allowing a highly efficient expression and minimising
possible negative effects on the viability and growth of the host
cells.
[0041] When, for example, the methylotrophic yeast Pichia pastoris
is used, the integrative can be incorporated into the yeast's
genome after transformation of the host, resulting in gene-tical
stability of the transformants (loss of plasmids is then of no
importance). It is possible to generate transformants with the
heterologous target gene under the control of e.g. the alcohol
oxidase (AOX) promotor), in which the recombinant gene is either
incorporated into the HIS4 locus or the AOX1 locus.
[0042] To ensure production of a non cleaved sequence a process
according to the invention for producing recombinant collagen like
material comprises use of a nucleic acid sequence encoding
recombinant collagen amino acid sequence substantially free of
protease cleavage sites of protease active in the expression host
cell. In the case of Pichia pastoris for example and possibly also
for other host cells a nucleic acid sequence encoding collagen of
which the corresponding amino acid sequence is free of
[Leu/Ile/Val/Met]-Xaa-Yaa-Arg wherein Xaa and Yaa correspond to Gly
and Pro or other amino acids and at least one of the amino acids
between the brackets is amended could be preferred.
[0043] The process suitably provides expression leading to peptide
harvest exceeding 2 g/liter or even exceeding 3 g/liter. The
process can suitably be carried out with any of the recombinant
collagen-like polypeptides defined above for the emulsion according
to the invention. Multicopy transformants can provide more than 14
grams of gelatin per liter of clarified broth at a biomass wet
weight of 435 grams per liter. Most suitably the product resulting
from microbial expression is isolated and purified until it is
substantially free of other protein, polysaccharides and nucleic
acid. As is apparent from the examples numerous methods are
available to the person skilled in the art to achieve this. The
process according to the invention can provide the expression
product isolated and purified to at least the following degree:
content nucleic acid less than 100 ppm, content polysaccharides
less than 5%, content other protein less than in commercial
products. More preferably the DNA content of less than 1 ppm, RNA
content less than 10 ppm even less than 5 ppm and polysaccharide
content less than 0.5% or even less than 0.05% can be achieved.
[0044] The invention also concerns a process of producing an
amphiphilic polymer in the manner described above, comprising
introducing a gene encoding an amphiphilic polypeptide part of said
polymer into a suitable host, culturing said host under conditions
suitable for expression of said gene, and recovering said
polypeptide. If desired the polypeptide can be coupled with another
peptide or non-peptide, natural or synthetic polymer to produce a
hybrid polymer suitable as an emulsifier.
[0045] In a preferred embodiment of the invention the gelatin-like
material comprises no cysteine residues. The presence of cysteine
in photographic product will disturb the product manufacturing
process. It is thus preferred that cysteine is present in as small
an amount as possible. Suitably photographic applications will
employ material comprising less than 0.1% cysteine.
EXAMPLES
[0046] The production of gelatin 1 (MW=54 kD) and gelatin 2 (MW=28
kD), which can be used in the emulsions of the invention, is
described in van Heerde et al. U.S. Pat. No. 6,150,081.
[0047] These gelatins are referred therein as COLIA1-2 and
COLIA1-1, respectively, and they are produced by transforming
Pichia pastoris with mouse COLIA1 gene and expressing the gene by
fermentation of the transformant Pichia strain.
Example 1
[0048] In this example the emulsification of current
state-of-the-art gelatins A and B was compared with the invention
(recombinant) gelatin 1 at pH=5.0 The average molecular weight of
the de-ionised lime bone gelatin A and a hydrolysed gelatin B were
respectively 177.4 and 23 kD, while the average molecular weight of
the invention (recombinant) gelatin 1 was 54 kD.
[0049] The average molecular weight was measured via GPC analysis,
the GPC method was carried out at 214 nm while the separation was
performed over 300*7.8 mm column (TOSO Haas) loaded with TSK-gel
4000 SWXL, the eluent consisted of 1 wt. % SDS, 0.1 mol/l
Na.sub.2SO.sub.4 and 0.01 mol/l NaH.sub.2PO.sub.4, at a flow of 0.5
ml/min.
[0050] The basic recipe of each emulsion batch (in a total volume
of 500 ml) contained 15 g gelatin, 43 g tricresyl phosphate TCP oil
and 435 gram of water. A surfactant amount of 0.4868 mM SDBS per 5
g gelatin per litre was added.
[0051] First the gelatin was dissolved in the necessary amount of
water and pH was adjusted to the required pH of 5.0. After pH
adjustment the required amounts of SDBS (from a 500 mmol/l SDBS
stock solution) and TCP were added.
[0052] Emulsification was carried on 2 ml scale, therefore 2 ml
sample was transferred to a plastic tube with a 10 ml capacity.
[0053] Pre-emulsification took place at 40.degree. C. using a
vortex mixer, final emulsification was carried out using a Branson
Sonifier 250 ultra-soon emulsifier for 4 minutes.
[0054] For the ultra-soon emulsification a tip with 3 mm diameter
was placed 0.5-1.0 cm below the upper emulsion level. Mixing with a
too high severity and also higher (than 0.5 cm below the upper
emulsion level) tip position will cause foam and therefore
inefficient energy transfer from tip to solution.
[0055] Initial size of the emulsions was measured immediately after
emulsification, within 2 minutes, average size was measured via
turbidity at .lamda.=500 and .lamda.=600 nm in a Hewlett Packard
8452A diode array spectrophotometer.
[0056] With a refractive index of TCP (=1.552) and the ratio of the
turbidities at .lamda.=600 nm and .lamda.=500 nm the average
droplet size is calculated based upon the theory of Mie (described
in the reference list).
Initial Size (in Nm) at pH=5, Addition of 0.4868 mMol [SDBS]
Surfactant/5 g Gelatin/Litre
TABLE-US-00002 gelatin type 15.degree. C. 25.degree. C. 40.degree.
C. invention recombinant 318 303 198 gelatin 1 (54 kD) current
state-of-the-art >500 >500 450 gelatin A (177 kD) current
state-of-the-art >500 >500 280 gelatin B (23 kD)
[0057] This example shows that OW emulsions that are prepared with
the invention (recombinant) gelatin 1 have a smaller initial size
than OW emulsions prepared with traditional gelatins A and B at
several temperatures. An advantage of the invention recombinant
gelatin 1 is that they can be used for emulsion making at
temperatures below 40.degree. C., while very unstable emulsions are
prepared at these temperatures with the state-of-the-art gelatins.
No gelation occurs at temperatures below the 40.degree. C. with the
invention gelatin 1 because the proline is not hydroxylated while
the pI of 9 for the invention gelatin is much more deviating than
the state-of-the-art gelatins at pH=5. This temperature effect
results in that the temperature control of the emulsification
process is much easier and less critical. The `normal` setting
temperature of current state of the art gelatin is about 30.degree.
C.
Example 2
[0058] In this example the emulsion ageing stability of current
state-of-the-art gelatins A and B was compared at pH=6.0 with the
invention (recombinant) gelatin 1 which has an average molecular
weight of 54 kD.
[0059] The emulsion preparation and emulsification conditions were
comparable to example 1. The only difference was that we measured
size stability in time, this means that turbidity at .lamda.=500
and at .lamda.=600 nm was measured after 0 h, 1 h, 2 h, 3 h and 4 h
after the emulsion was prepared. The size difference between 4
hours ageing and 0 hour ageing was plotted in the table below.
Ageing Properties of Recombinant Gelatin Compared to Traditional,
State of the Art, Gelatin, pH 6.0, 0.4868 mMol [SDBS]/5 g
Gelatin/L. The Size Difference is Defined as the Difference in Size
(in Nm) Between 4 and 0 Hours (Fresh) of Ageing.
TABLE-US-00003 15.degree. C. 25.degree. C. 40.degree. C. invention
recombinant 100 159 158 nm gelatin 1 (54 kD) current
state-of-the-art >500 >500 247 nm gelatin A current
state-of-the-art >500 190 200 nm gelatin B
[0060] This example shows that the size stability of the OW
emulsion stabilised with recombinant gelatin 1 is better than the
stability of an OW emulsion stabilised with traditional current
state-of-the-art gelatins A and B.
[0061] From the example it is clear that a temperature decrease of
the OW emulsion to 15.degree. C. increases the size stability
significantly with the invention (recombinant) gelatin 1. This
temperature decrease can not be realised with the traditional
gelatins because gelation happens. So the absence of gelation makes
a temperature shift in the production process possible (e.g.
transfer of the emulsion to a "waiting tank" to increase the size
stability before use in the "final emulsion").
[0062] The same results were visible at pH 5.0; the invented
recombinant gelatin gives due to its higher pI more flexibility in
the pH of the emulsification process.
Example 3
[0063] In this example traditional gelatin A and recombinant
gelatin 1 are mixed in various ratio's, to show the possibility
that not only recombinant gelatin but also mixtures of recombinant
and traditional gelatins can be used.
[0064] The mixtures were prepared by mixing the proper ratios of
solutions of traditional and recombinant gelatin before
emulsification.
[0065] Emulsion preparation, emulsification conditions and size
measurements were carried out in the same way as described in
examples 1 and 2.
Test Conditions: pH=6.0, 40.degree. C., Addition of 0.4868 mMol
[SDBS]/5 g Gelatin/Litre. The Size Ageing is Defined as the
Difference in Size (in Nm) Between 4 and 0 Hours (Fresh)
Ageing.
TABLE-US-00004 Ageing 0-->4 h invented recombinant gelatin 1 (54
kD) 158 nm mix recombinant 54 kD/current gelatin 222 nm A in ratio
1/1 mix recombinant 54 kD/current gelatin 218 nm A in ratio 1/3
current state-of-the-art gelatin A 247 nm
[0066] This example shows that the improved size stability of the
OW emulsion can also be obtained with mixtures of the invented
(recombinant) gelatins and the traditional gelatin A.
[0067] The stability increase of the mixtures is less pronounced
compared to the stability of only recombinant gelatin however
improved stability is still visible.
Example 4
[0068] In this example the emulsion size stability after ageing for
4 hours of current state-of-the-art gelatin A was compared with the
invention recombinant gelatin 1 at various surfactant
[SDBS]-concentrations and at pH=6.0.
[0069] The emulsions are prepared in the same way as described in
the examples 1, 2 and 3, the only difference is that the [SDBS] is
adjusted to the required [SDBS] by adding more amount of SDBS from
a 500 mmol/1 [SDBS] stock solution.
[0070] Emulsification and size measurements in time are also
measured in the same way as described in the previous mentioned
examples.
[0071] Ageing at various [SDBS] at pH 6.0 and 40.degree. C. The
difference is defined as the difference between the size after 4
and after 0 hours ageing.
TABLE-US-00005 [SDBS] mmol/5 g recombinant current state-of-the-
gelatin/litre gelatin 1 (54 kD) art gelatin A 0.064 245 280 nm
0.4868 158 247 nm 0.854 240 263 nm 3.5 360 370 nm 20 430 410 nm
[0072] The improved stability of the invented (recombinant) gelatin
is clearly visible at [SDBS] below 1 mmol/l/5 g gelatin, above this
concentration the gelatin concentration at the interface is
decreasing and is therefore less important in stability.
[0073] These results indicate that the same or even improved
stability for recombinant gelatin compared to traditional gelatin
can be obtained at lower [SDBS], see the single FIGURE of the
accompanying drawing.
Example 5
[0074] In this example the initial size of emulsions prepared with
current state-of-the-art gelatin A was compared at pH 5.0 with two
invention (recombinant) gelatins 1 and 2 with average molecular
weights of respectively 54 and 28 kD. The preparation of the
emulsions, but also the size measurements was done in the same way
as described in example 1.
Initial Size of Emulsions Prepared with Different Recombinant
Molecular Weight Gelatin. Emulsion pH=5.0; [SDBS]=0.4868 mM/5 g
Gelatin/Litre
TABLE-US-00006 samples initial size (in nm) Invention recombinant
gelatin 3 (57 kD 150 with amphipolar character Invention
recombinant gelatin 1 (54 kD) 198 Invention recombinant gelatin 2
(28 kD) 290 Current state of the art gelatin A 450
[0075] This experiment shows that the initial size of the OW
emulsion is significantly improved when recombinant gelatins are
applied already with a molecular weight of 28 kDa. Invented
gelatins with a higher molecular weight of 54 kDa enables further
improvement for the initial size of the OW emulsion
[0076] The lowest initial size has been realised with the invented
(recombinant) gelatin with a MW of 57 kDa, which has an amphipolar
bi-functional character. This bi-functional character is obtained
by a collagen, which is synthetically made from two a-polar
building blocks (called N1 and N2) and one polar block (called P1).
The blocks are combined as N1N2P1P1P1P1 such that the total polar
P-"leg" sticks into the water-phase and the apolar N1N2-"leg" is
adsorbed at the oil-interphase. The lowest initial size of the
OW-emulsion has been achieved with this new developed bi-functional
collagen. The synthesis route of this invented gelatin with an
amphipolar character is described in the following text.
Example 6
Materials and Methods and Analysis of Bipolar Recombinant
Collagen-Like Polymer
General Molecular-Biological Techniques
[0077] Cloning procedures were performed essentially according to
Maniatis et al. [1]. Plasmid DNA was isolated using Wizard Plus SV
miniprep, or Qiagen midiprep systems. DNA was isolated from agarose
gels using the QIAquick Gel Extraction Kit (Qiagen). All enzymes
used were from Amersham Pharmacia Biotech unless otherwise stated
and were used according to the manufacturer's recommendations. All
procedures involving the handling and transformation of Pichia
pastoris were essentially performed according to the manual of the
Pichia Expression Kit (Invitrogen) [2].
Construction of pPIC9-N1N1P4 and pPIC9-N1N2P4
[0078] Custom-designed bipolar gelatins were constructed by
combining polar and nonpolar modules. Each module has a molecular
weight of approximately 10 kDa. The design is such that, in
principle, any combination and number of modules can be combined in
any order desired. Molecules consisting of two nonpolar modules and
four polar modules (P) were constructed. One molecule (N1N1P.sub.4)
contains two identical nonpolar modules (N1). Another molecule
(N1N2P.sub.4) contains two different nonpolar modules (N1 and N2).
The N2 module is similar to the N1 module, but differs mainly in
the presence of a cluster of methionine and charged residues at its
C-terminal side.
[0079] The polar gelatin module (P monomer) was constructed as
described in van Heerde et al. U.S. Pat. No. 6,150,081, where it is
used in base emulsion applications [3]. The gene was designed to
have the codon usage of Pichia pastoris highly expressed genes
(Sreekrishna and Kropp [4]). Two separate PCR reactions were
performed, using the following oligonucleotides: [0080] 1. 1 pmol
OVL-PA-FW, 1 pmol OVL-PA-RV, 50 pmols HLP-PA-FW and 50 pmols
HLP-PA-RV. [0081] 2. 1 pmol OVL-PB-FW, 1 pmol OVL-PB-RV, 50 pmols
HLP-PB-FW and 50 pmols HLP-PB-RV.
[0082] The 50 .mu.l PCR reactions were performed in a GeneAmp 9700
(Perkin-Elmer) and contained 0.2 mM dNTP's (Pharmacia), 1.times.Pwo
buffer (Eurogentec) and 1.25 U Pwo polymerase (Eurogentec).
Reaction 1 involved 18 cycles consisting of 15 seconds at
94.degree. C. and 15 seconds at 72.degree. C. Reaction 2 involved a
touchdown PCR, whereby each cycle consisted of 15 seconds at
94.degree. C., 15 seconds at the annealing temperature and 15
seconds at 72.degree. C. The annealing temperature was lowered from
72.degree. C. to 68.degree. C. in the first 5 cycles, after which
20 additional cycles at an annealing temperature of 67.degree. C.
were performed.
[0083] The PCR products were isolated from agarose gel. 0.3 pmols
of each fragment and 50 pmols of the outer primers HLP-PA-FW and
HLP-PB-RV were subjected to overlap extension PCR. 25 cycles
consisting of 15 seconds at 94.degree. C., 15 seconds at 67.degree.
C. and 15 seconds at 72.degree. C. were performed. The resulting
0.3 kb PCR fragment was digested with XhoI/EcoRI and inserted in
cloning vector pMTL23. An errorless clone was selected by
verification of the sequence by automated DNA sequencing.
[0084] In order to create a P tetramer for the polar part of the
bipolar gelatins, the P module was released by digesting the vector
with DraIII/Van91I. In a separate reaction the vector was digested
with Van91I and dephosphorylated. The DraIII/Van91I fragment was
then inserted into this Van91I digested vector. This yielded a
vector containing a P dimer. This dimer was released by digestion
with DraIII/Van91I and reinserted into the Van91I site of the dimer
bearing vector, yielding pMTL23-P4.
[0085] Analogous to the construction of the polar P monomer
gelatin, two different nonpolar gelatins N1 and N2, respectively,
were constructed. The genes were designed to have the codon usage
of P. pastoris highly expressed genes (Sreekrishna and Kropp [4]).
Two separate reactions were performed for both N1 and N2, using the
following oligonucleotides:
N1:
[0086] 1. 1 pmol OVL-NA-FW, 1 pmol OVL-N1A-RV, 50 pmols HLP-PA-FW
and 50 pmols HLP-N1A-RV. [0087] 2. 1 pmol OVL-N1B-FW, 0.1 pmol
OVL-N1B-RV, 50 pmols HLP-N1B-FW and 50 pmols HLP-PB-RV.
N2:
[0087] [0088] 3. 1 pmol OVL-NA-FW, 1 pmol OVL-N2A-RV, 50 pmols
HLP-PA-FW and 50 pmols HLP-N2A-RV. [0089] 4. 1 pmol OVL-N2B-FW, 1
pmol OVL-N2B-RV, 50 pmols HLP-N2B-FW and 50 pmols HLP-PB-RV.
[0090] Reaction conditions for N1 and N2 module reactions 1, 3 and
4 were as for P monomer reaction 2. The first 5 cycles of N1 module
reaction 2 consisted of 15 seconds at 98.degree. C. and 15 seconds
at 72.degree. C. without presence of primers HLP-N1B-FW and
HLP-PB-RV. These primers were then added and 20 cycles consisting
of 15 seconds at 94.degree. C. and 15 seconds at 72.degree. C. were
performed.
[0091] The PCR products were purified from agarose gel and overlap
extension PCR was performed using 0.3 pmols of each fragment and 50
pmols of the outer primers HLP-PA-FW and HLP-PB-RV. Each PCR cycle
consisted of 15 seconds at 94.degree. C., 15 seconds at the
annealing temperature and 15 seconds at 72.degree. C. The annealing
temperature was lowered from 72.degree. C. to 68.degree. C. in the
first 5 cycles, after which 20 additional cycles at an annealing
temperature of 67.degree. C. were performed. The resulting 0.3 kb
PCR fragments were digested with XhoI/EcoRI and inserted in cloning
vector pMTL23, yielding vectors pMTL23-N1 and pMTL23-N2. Errorless
clones were selected by verification of the sequence by automated
DNA sequencing.
[0092] Vector pMTL23-N1 was digested with DraIII and
dephosphorylated, after which one DraIII/Van91I digested N1 module
was inserted to form pMTL23-N1N1. Likewise, one DraIII/Van91I
digested module was inserted in DraIII digested and
dephosphorylated vector pMTL23-N2 to form vector pMTL23-N1N2. The
N1N1 and N1N2 modules were released by digestion with DraIII/Van91I
and were ligated into DraIII digested and dephosphorylated
pMTL23P.sub.4 to yield constructs pMTL23-N1N1P.sub.4 and
pMTL23-N1N2P4, respectively. The resulting N1N1P.sub.4 and
N1N2P.sub.4 inserts were then released by digestion with XhoI/EcoRI
and cloned in the XhoI/EcoRI sites of Pichia expression vector
pPIC9. The encoded amino acid sequence of the mature (processed)
N1N1P.sub.4 and N1N2P.sub.4 gelatins are provided in the sequence
listing that follows:
N1N2P.sub.4 has a theoretical molecular weight: 57 kD, isoelectric
point: 5.8 N1N1P.sub.4 has a theoretical molecular weight: ca 57
kD, isoelectric point: 4.9 Transformation of Pichia pastoris with
pPIC9-N1N1P4 and pPIC9-N1N2P4
[0093] In order to obtain Mut.sup.+ transformants upon
transformation of P. pastoris (i.e. fast-growing on methanol), the
constructs were linearized with SalI in order to target integration
of the construct into the his4 gene, keeping the AOX1 locus intact
[2]. It will be understood that Mut.sup.S transformants (i.e.
slow-growing on methanol) can in principal also be used, but
Mut.sup.+ was chosen for practical reasons.
[0094] After phenol extraction and ethanol precipitation, the
construct was then used to transform P. pastoris strain GS115
(Invitrogen) using electroporation according to Becker and Guarente
[5] using the BioRad GenePulser (set at 1500V, 25 .mu.F and 20052
and using 0.2 cm cuvettes). The transformation mix was plated out
on Minimal Dextrose plates (MD-plates; 1.34% YNB,
4.times.10.sup.-5% biotin, 1% dextrose and 1.5% agar) in order to
select for the presence of the vector which converts the His.sup.-
strain GS115 to His.sup.+. After growth at 30.degree. C. for 3
days, several colonies were selected for PCR confirmation of the
Mut.sup.+ genotype. The PCR machine used was the Perkin-Elmer
GeneAmp 9700. Colony PCR was performed using 50 pmol 5'AOX1 primers
Seq id nr. 24, 50 pmol 3'AOX1 primer Seq id nr 25, 1.25 U Taq
polymerase (Pharmacia), 0.2 mM dNTPs (Pharmacia) and 1.times.Taq
buffer (Pharmacia) in a total volume of 50 .mu.l. After an initial
denaturation at 94.degree. C. for 5 minutes, 30 cycles were
performed consisting of 15 seconds at 94.degree. C., 30 seconds at
57.degree. C. and 2 minutes at 72.degree. C. Final extension was at
72.degree. C. for 10 minutes. Agarose gel electrophoresis should
reveal a 2.2 kb endogenous AOX1 band for Mut.sup.+
transformants.
Production of N1N1P.sub.4 and N1N2P.sub.4
[0095] Selected transformants were fermented in fed-batch mode
according to the Pichia fermentation guidelines of Invitrogen.
Cells were grown in a 1-litre fermentor (Applikon) in the initial
experimental stages to optimise protein production. Thereafter
cells were grown in a 20-litre or a 140-litre fermentor (Biobench
20, Bio-pilot 140, Applikon) for pilot scale production of gelatin.
Working volumes were 1-litre, 15-litre and 100-litre, respectively.
AD1020 controllers (Applikon) were used to monitor and control the
fermentation parameters. The program BioXpert (Applikon) was used
for data storage. Dissolved oxygen levels were monitored in the
fermentor using an oxygen electrode (Ingold for 1-litre
fermentations, Mettler Toledo for larger scale fermentations).
Agitation (500-1000 rpm) and aeration (1-2 vvm, i.e. 1-2
LL.sup.-1min.sup.-1) were manually adjusted to keep the dissolved
oxygen concentration above 20%. pH was measured by a pH electrode
(Broadly James cooperation) and automatically kept at pH 3.0 by
addition of ammonium hydroxide (25%), which also served as nitrogen
source for growth of the micro organisms. An anti foam-electrode
was used to prevent excessive foaming. When necessary, the anti
foam Structol J673 (Schill and Seilacher, Hamburg, Germany) or the
organic anti foam 204 (from Sigma_Aldrich, Bornem, Belgium) was
used. Growth of the micro-organisms was monitored by determination
of the cell dry weight. A calibration curve was made by means of
which cell wet weight could be converted into cell dry weight. Cell
wet weight was determined after centrifugation of 2 ml samples for
5 min at 15.000 rpm and removing the supernatant. Cell dry weight
was determined after addition of 200 .mu.l of cells to a pre-dried
filter (0.45 .mu.m membrane, Schleicher & Schiill, Dassel,
Germany), washing with 25 ml of deionized water and drying in a
microwave oven for 15 minutes at 1000 W. Cell dry weight was
approximately a factor 3 lower than cell wet weight. Precultures
were started from colonies on a MGY plate, in flasks containing a
total of 10% of the initial fermentation volume of MGY. The volume
of the medium was 20% of the total flask volume. Cells were grown
at 30.degree. C. at 200 rpm in a rotary shaker for 24-60 hours. The
fermentation basal salts medium in the fermentor contained per
liter: 26.7 ml of phosphoric acid (85%), 0.93 g calcium sulphate,
18.2 g potassium sulphate, 14.9 g magnesium sulphate.7H.sub.2O,
4.13 g potassium hydroxide and 40.0 g glycerol. An amount of 4.3 ml
of PTM.sub.1 trace salts was added per litre of fermentation basal
salts medium.
[0096] PTM.sub.1 trace salts contained per litre: 4.5 g cupric
chloride.2H.sub.2O, 0.09 g potassium iodide, 3.5 g manganese
chloride.4H.sub.2O, 0.2 g sodium molybdate.2H.sub.2O, 0.02 g boric
acid, 1.08 g cobalt sulphate.7H.sub.2O, 42.3 g zinc
sulphate.7H.sub.2O, 65.0 g ferrous sulphate.7HO, 0.2 g biotin and
5.0 ml sulphuric acid. Trace salts were filter sterilised.
[0097] The fermentor was sterilised with the fermentation basal
salts medium. The 20-litre and 120-litre fermentor were sterilised
in situ with initial medium volumes of 5-7.5 1 and 50-litre,
respectively. The 1-litre fermentor was sterilised with 500 ml
medium in an autoclave. After sterilisation the medium was
supplemented with sterile 1% casamino acids (optional).
[0098] The temperature was set at 30.degree. C., agitation and
aeration were set at 500 rpm and 1 vvm (i.e. 1
LL.sup.-1min.sup.-1), respectively. The pH was adjusted to set
point (pH 5.0) with 25% ammonium hydroxide. Trace salts were
aseptically added to the medium. The fermentor was inoculated with
10% of the initial fermentation volume of precultured cells in MGY.
The batch culture was grown until the glycerol was completely
consumed (18-24 hours). This was indicated by an increase of the
dissolved oxygen concentration to 100%. Cell dry weight was 25-35
g/l in this stage. Thereafter the glycerol fed-batch phase was
started by initiating a 50% (v/v) glycerol feed containing 12 ml
PTM.sub.1 trace salts per litre of glycerol. The glycerol feed was
set at 18 ml/h/litre initial fermentation volume. The glycerol feed
was carried out for 4 hours, or overnight in the case of a long lag
phase. During the glycerol batch phase the pH of the fermentation
medium was lowered to 3.0.
[0099] An additional 1% of casamino acids were added (optional)
after which the protein induction phase was initiated by starting a
100% methanol feed containing 12 ml PTM.sub.1 trace salts per liter
of methanol. The feed rate was set to 3 ml/h/litre initial
fermentor volume. During the first hours methanol accumulated in
the fermentor. After 2-4 hours dissolved oxygen levels decreased
due to adaptation to methanol. The methanol feed was increased to 6
ml/h/initial fermentor volume in the case of a fast dissolved
oxygen spike. If the carbon source is limiting, shutting off the
carbon source causes the culture to decrease its metabolic rate and
the dissolved oxygen concentration to rise (spike). After an
additional 2 hours the methanol rate was increased to 9 ml/h/litre
initial fermentor volume. This feed rate was maintained throughout
the remainder of the fermentation. The fermentation was stopped
after 70-130 h methanol fed-batch phase. During the fermentation
samples were taken of 2 ml, centrifuged (5 min, 15.000 rpm) and the
supernatant was stored at -20.degree. C.
[0100] At the end of the fermentation, the cells were removed by
centrifugation (10.000 rpm, 30 min, 4.degree. C.), followed by
micro filtration (cut off 0.2 .mu.m) in the case of the 1-litre
fermentation. Cells were removed by micro filtration in the case of
the 20- or 100-litre fermentation.
[0101] In the case of 20-L fermentation, the cell broth was applied
to a microfiltration module containing a poly ether sulphone
membrane with 0.20 .mu.m pore size (type MF 02 M1 from X-Flow,
fitted in a Rx 300 filtration module from X-Flow). In the case of
the 100-liter fermentation cells were removed by a pilot plant
scale cross flow micro filtration unit containing a hollow fiber
poly ether sulphone membrane with 0.2 .mu.m pore size (type MF 02
M1, from X-Flow, fitted into a R-10 membrane module). These
filtration units are mentioned merely as examples. It will be
understood that any suitable micro filtration system could be
applied to remove the cells. Optionally, the bulk of cells and
debris was removed by centrifugation, and only the supernatant and
the medium used to wash the cells was applied to the micro
filtration units.
[0102] In the case of the 100 Litre fermentation, the cell broth
was first applied to a filtration unit fitted with a stack of flat
0.4.times.0.4 m cellulose Bio 10 or Bio 40 depth filters (USF Seitz
Filter Werke, Bad Kreutznach, Germany) with a total filtration
surface of 1.9 m.sup.2. Thereafter, the permeate was filtered with
a spiral wound 0.2 .mu.m pore size poly sulphone dead end micro
filtration unit (USF Seitz Filter Werke, Bad Kreutznach, Germany).
After micro filtration, the filters were sterilised, the cells were
destroyed by steam sterilisation or by autoclaving, and the absence
of recombinant Pichia cells in the filtrate was verified by plating
out samples of filtrate.
Purification of Synthetic Gelatins from the Cell Free Fermentation
Broth
Separation of Recombinant Bipolar Gelatins and Non-Recombinant
Pichia Proteins or Small Peptides (Optional).
[0103] For separation of recombinant bipolar gelatins and
non-recombinant Pichia proteins, cell-free fermentation broth was
subjected to differential precipitation (=fractionation) at 40-80
volume-% ethanol or acetone. At 40 volume-% ethanol or acetone, the
non-gelatinous proteins (from Pichia) were precipitated, while at
60-80 volume-% ethanol or acetone, gelatin was precipitated, as
shown by SDS-PAGE and analysis of the amino acid composition. Small
peptides and other low molecular weight contaminants remained in
solution at 80 vol.-% ethanol or acetone. Ethanol or acetone was
cooled for 2-4 hours at .+-.20.degree. C. An amount of 40 vol.-% of
ice-cold ethanol or acetone (v/v) was added slowly to the
pre-cooled supernatant from the fermentation at 4.degree. C. under
magnetic stirring. Supernatant was stirred overnight at 4.degree.
C. Precipitated proteins and particles were removed by
centrifugation (4.degree. C., 10.000 rpm, 30 min). The pellet was
resuspended in 40 vol.-% ice-cold ethanol or acetone and again
centrifuged. Both 40 vol.-% acetone supernatant fractions were
pooled. Thereafter the supernatant was brought to 60-80 vol.-%
ethanol, or acetone (v/v) and stirred overnight. Precipitated
proteins were collected by centrifugation. The pellet was dissolved
in an appropriate amount of water. In addition to ultra filtration
or evaporation of water, precipitation of gelatin at 80% (70-90%)
ethanol or acetone can also be used to concentrate the protein.
Purification of Recombinant Gelatin from Fermentation Broth by
Anion Exchange Chromatography.
[0104] At laboratory (mg to g) scale, the recombinant gelatin was
optionally captured and purified from the fermentation broth by
anion exchange chromatography (e.g. using Q Sepharose HP or XL from
Amersham Pharmacia Biotech, Uppsala, Sweden), preferably in 20 mM
phosphate, carbonate or borate buffer at pH 6 to 8 and using a NaCl
gradient or step gradient for elution.
Purification of Recombinant Gelatin from Cell Free Fermentation
Broth by Ammonium Sulphate Precipitation.
[0105] The recombinant gelatin was purified from non-recombinant
proteins and peptides, polysaccharides, nucleic acids and other
contaminating molecules by selective precipitation of the gelatin
at 60% saturation of ammonium sulphate. Ammonium sulphate was
slowly added to 60% saturation at 4.degree. C. After 60 min
stirring the sample was centrifuged (30 min, 4.degree. C., 10,000
rpm). The pellet was resuspended in 60% ammonium sulphate and again
centrifuged. If more than 1% (w/w) polysaccharides or sugars
remained present, the ammonium sulphate precipitation procedure
described above was repeated after complete redissolving of the
gelatin in the absence of ammonium sulphate.
[0106] Alternatively, the ammonium sulphate precipitate was
collected on a suitable depth filter (e.g. Bio 10, Bio 40, or AKS5
from USF Seitz Filter Werke, Bad Kreuznach, Germany) and washed
free from contaminating components by flushing the filter and
filter cake with 60-70% ammonium sulphate.
[0107] Finally, the purified gelatin pellet or filter cake was
dissolved in de-ionised water.
[0108] At laboratory scale, milligram to gram quantities of
purified recombinant gelatin were desalted by dialysis against
deionised water, which was refreshed every 4 hours. Dialysis
membranes of regenerated cellulose (Spectra Por.RTM., from
Spektrum) were used with a molecular weight cut-off of 8 kD. The
dialysis was stopped after 2-7 days when the electrical
conductivity of the sample was judged to be sufficiently low.
[0109] At pilot scale (i.e. gelatin quantities of more than 10 to
100 gram), the purified recombinant gelatin was desalted by
ultrafiltration/diafiltration, using poly ether sulphone membranes
with a molecular cut-off of 4 (1 to 10) kD. The
ultrafiltration-diafiltration was stopped when the electrical
conductivity of the gelatin solution was sufficiently low. This
typically occurred after 10 to 30 cycles of dilution (2.times.) and
concentration (2.times.).
[0110] Optionally, an additional, final desalting step was carried
out by adding a slight excess of mixed-bed ion exchange resin beads
(e.g. Amberlite MB-3 from Merck, Darmstadt, Germany) and incubating
for 30 min. to maximally 1 hour.
[0111] Conductivity was measured with a digital conductivity meter
(Radiometer), calibrated with 1 mM and 10 mM KCl solutions (140 and
1400 .mu.S.cm.sup.-1, respectively). After desalting by dialysis or
diafiltration, the final electrical conductivity was typically 5 to
15 .mu.S.cm.sup.-1.((gram gelatin).L.sup.-1).sup.-1. After further
desalting with mixed bed ion exchange beads, the final electrical
conductivity was typically 0.5 to 3 .mu.S.cm.sup.-1.((gram
gelatin).L.sup.-1).sup.-1.
[0112] Where applicable, the product was pre-dried (optional) by
precipitation with high concentrations of acetone and evaporation
of the acetone.
[0113] The purified and desalted product was either freeze-dried or
spray-dried.
Characterisation of the Gelatin Product
Molecular Weight Distribution and Contaminating Proteins
[0114] The molecular weight distribution of the recombinant gelatin
product and the presence of contaminating proteins were analyzed by
denaturing poly acrylamide gel electrophoresis (SDS-PAGE) [6] in a
Mini-PROTEAN II system (from Biorad) and Coomassie Brilliant blue
staining. Optionally, a higher voltage was applied (400 Volt), in
order to speed-up the electrophoresis rate and minimize protein
diffusion. For both bipolar gelatin types a single gelatin band was
observed. After purification as described above (e.g. micro
filtration, ammonium sulphate fractionation and desalting), the
product was apparently homogeneous and free from contaminating
proteins.
[0115] The molecular weight apparent from SDS-PAGE, as calibrated
with globular protein molecular weight markers, was too high, in
accordance with numerous other observations on gelatins and
collagen-like proteins and polypeptides.
[0116] However, after gel filtration of the ammonium
sulphate-purified gelatin with a Superose-12 column (Amersham
Pharmacia Biotech, Uppsala, Sweden), a single gelatin peak eluted
at the correct theoretical molecular weight, with reference to a
set of 6 distinct fragments of recombinant mouse type I collagen,
having known (i.e. theoretical and experimentally verified)
molecular weights of 8, 12, 16, 28, 42 an 54 kDa. The gelatin
samples were eluted from the Superose-12 column with 100 mM NaCl
and a flow of 0.2 mL/min. This showed that the recombinant gelatin
product was correctly expressed and was not degraded to any
considerable extent. The correctness of molecular weight was
confirmed by mass spectrometry.
Confirmation of N-Terminal Amino Acid Sequence
[0117] After SDS-PAGE as described above, the proteins in the gel
were blotted onto an Immobilon P.sup.SQ membrane (from Millipore)
by applying 100 V for one hour in a Mini Trans-Blot Cell (Biorad).
Transfer buffer was 2.2 g CAPS per litre of 10% methanol, pH 11.
Blots were stained with Coomassie Brilliant Blue and selected bands
were cut out. N-terminal protein sequencing was performed by Edman
degradation. It appeared that the N-terminal sequence of both
recombinant gelatin products was correct.
Confirmation of Purity and Amino Acid Composition
[0118] The amino acid composition of the purified gelatin product
was determined after complete HCl-mediated hydrolysis of the
peptide bonds at elevated temperature, followed by derivatisation
of the amino acids with a fluorophore, and HPLC.
[0119] The percentage Gly expected from pure gelatin is 33%. This
offers a means of estimating the purity of produced recombinant
gelatins. In order to correct for the percentage of Gly in
endogenously secreted proteins of P. pastoris, amino acid
composition analysis was performed on fermentation supernatant of a
Mut.sup.+ trans-formant of pPIC9. The percentage Gly found was 9%.
The purity of a sample can now be estimated by the formula:
(% Gly-9)/(33-9)=(% Gly-9)/24.
Determination of Contamination with Polysaccharides, Sugars and
Nucleic Acids
[0120] After dissolution of samples in MilliQ water, the following
assays were performed. The sugar content was determined by a
phenol-based assay. 200 .mu.L samples were mixed with 200 .mu.l 5%
(w/w) phenol. After thorough mixing using a Vortex mixer, 1 mL of
concentrated sulphuric acid was added. After mixing, the samples
were incubated for 10 min at room temperature and, subsequently,
for 20 min at 30.degree. C. After cooling, the light absorption of
the samples at 485 nm was determined. Analytical grade mannose was
used to prepare the calibration curve.
[0121] The DNA content was determined by mixing aliquots of diluted
SYBR.RTM. Green I nucleic acid `gel` stain (10.000.times. conc. In
DMSO) from Molecular Probes with our samples. After thorough
spectral analysis, the excitation wavelength was chosen to be 490
nm, and the emission wavelength 523 nm. The calibration was by
subsequent addition of known amounts of DNA to this same mixture,
as internal standards. Thus, a calibration curve was constructed.
Furthermore, it was checked that subsequent addition of
DNA-degrading enzyme resulted in complete break down of the
fluorescent signal.
[0122] A quantitative indication of the RNA plus DNA-content was
subsequently obtained by using SYBR.RTM. Green II `RNA gel stain`,
instead of SYBR.RTM. Green I. After thorough spectral analysis, the
excitation wavelength was chosen to be 490 nm, and the emission
wavelength 514 nm. Calibration was by subsequent addition of known
amounts of RNA. The resulting value was pronounced to be the `RNA`
content of the sample. In the absence of DNA, it corresponded to
the true RNA content. When present, the DNA-associated fluorescence
may have biased the RNA values, although a final addition of RNAse
was used to discern the DNA- and RNA-derived contributions to the
fluorescence.
Quantification of the Recombinant Product
[0123] The protein content was determined by the BCA assay from
Pierce, using gelatin from Merck (Darmstadt, Germany) as a
reference. The gelatin content of the cell-free fermentation broth
and various semi-purified gelatin samples was determined by
fractionating the samples at 40 and 80 volume-% of ethanol or
acetone, as described above, and quantifying the protein content of
the 40% pellet (i.e. non-recombinant Pichia protein), the pellet
obtained by enhancing the solvent content of the 40% supernatant to
80 vol.-% (i.e. precipitated recombinant gelatin), as well as the
80% supernatant (small peptides and bias from other molecules).
Again the BCA assay from Pierce was applied, using gelatin from
Merck as a reference. The purified and dried product was in
addition quantified by weight determination.
RESULTS
Gelatin Batch Produced at Laboratory Scale
Example
[0124] about 1 gram [0125] purification: micro filtration,
(NH.sub.4).sub.2SO.sub.4, desalting by dialysis, lyophilisation.
[0126] DNA: 2 ppm (w/w) [0127] RNA: 10 ppm (w/w) [0128] total
sugars: 1% (w/w) [0129] purity calculated from amino acid
composition determination conductivity: 5 .mu.S.cm.sup.-1.((gram
gelatin).L.sup.-1).sup.-1 gelatin was single-component according to
SDS-PAGE and FPLC.
REFERENCES CITED
[0129] [0130] [1] Maniatis T., Fritsch, E. F. & Sambrook, J.
(1982) Molecular cloning: A laboratory manual. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. [0131] [2] Manual of the
Pichia Expression Kit Version E (Invitrogen, San Diego, Calif.,
USA). [0132] [3] EP-A-0926432, NL-A-1007908 and EP-A-1014176, all
non-prepublished. [0133] [4] Sreekrishna, K. and Kropp, K. E.
(1996) Pichia pastoris, Wolf, K. (Ed), Nonconventional yeasts in
biotechnology. A handbook, Springer-Verlag, pp. 6/203-6/253. [0134]
[5] Becker, D. M. & Guarente, L. (1991) High efficiency
transformation of yeast by electroporation. Methods in Enzymology,
vol. 194: 182-187. [0135] [6] Laemmli, U. K. (1970) Cleavage of
structural proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680-685.
Sequence CWU 1
1
25126DNAArtificial SequencePrimer HLP-PA-FW 1gcgctcgaga aaagagaggc
tgaagc 262108DNAArtificial SequencePrimer OVL-PA-FW 2gcgctcgaga
aaagagaggc tgaagctggt ccacccggtg agccaggtaa cccaggatct 60cctggtaacc
aaggacagcc cggtaacaag ggttctccag gtaatcca 1083110DNAArtificial
SequencePrimer OVL-PA-RV 3tgagaacctt gtggaccgtt ggaacctggc
tcaccaggtt gtccgttctg accaggttga 60ccaggttgac cttcgtttcc tggttgacct
ggattacctg gagaaccctt 110424DNAArtificial SequencePrimer HLP-PA-RV
4tgagaacctt gtggaccgtt ggaa 24524DNAArtificial SequencePrimer
HLP-PB-FW 5ttccaacggt ccacaaggtt ctca 246115DNAArtificial
SequencePrimer OVL-PB-FW 6ttccaacggt ccacaaggtt ctcagggtaa
ccctggaaag aatggtcaac ctggatcccc 60aggttcacaa ggctctccag gtaaccaagg
ttcccctggt cagccaggta accct 1157108DNAArtificial SequencePrimer
OVL-PB-RV 7gcgtctgcag tacgaattct attagccacc ggctggaccc tggtttcctg
gtttaccttg 60ttcacctggt tgaccagggt tacctggctg accaggggaa ccttggtt
108826DNAArtificial SequencePrimer HLP-PB-RV 8gcgtctgcag tacgaattct
attagc 26926DNAArtificial SequencePrimer HLP-PA-FW 9gcgctcgaga
aaagagaggc tgaagc 2610111DNAArtificial SequencePrimer OVL-NA-FW
10gcgctcgaga aaagagaggc tgaagctggt ccacccggtg ttccaggttt cattggattc
60cctggtttgc caggatggcc aggtgtcttc ggtattcctg gttacccagg t
11111114DNAArtificial SequencePrimer OVL-N1A-RV 11tggccaacct
ggaaaaccag gccatcctgg gtaaccagga taaccgaaga tacctgggaa 60acctggccaa
ccaggccagc caaggtaacc tgggtaacca ggaataccga agac
1141225DNAArtificial SequencePrimer HLP-N1A-RV 12tggccaacct
ggaaaaccag gccat 251325DNAArtificial SequencePrimer HLP-N1B-FW
13atggcctggt tttccaggtt ggcca 2514107DNAArtificial SequencePrimer
OVL-N1B-FW 14atggcctggt tttccaggtt ggccaggatt cattggtctg cctggttact
tgggaccatg 60gggttttgtt ggttggcctg gttggttggg ttacccaggt ttgttcg
10715108DNAArtificial SequenceDPrimer OVL-N1B-RV 15gcgtctgcag
tacgaattct attagccacc ggctggaccg tggtcaccgg ggattccctc 60gtgaccaggg
taacctggta atccgaacaa acctgggtaa cccaacca 1081626DNAArtificial
SequencePrimer HLP-PB-RV 16gcgtctgcag tacgaattct attagc
2617106DNAArtificial SequencePrimer OVL-N2A-RV 17catagatacc
agggtaacca aatggtccca accaaccgaa aggtcctggc caacctggcc 60aaccaggcca
gccaaggtaa cctgggtaac caggaatacc gaagac 1061830DNAArtificial
SequenceDPrimer HLP-N2A-RV 18catagatacc agggtaacca aatggtccca
301930DNAArtificial SequencePrimer HLP-N2B-FW 19tgggaccatt
tggttaccct ggtatctatg 3020116DNAArtificial SequencePrimer
OVL-N2B-FW 20tgggaccatt tggttaccct ggtatctatg gttggccagg tttcctgggt
taccctggta 60tcttcggacc atggggtcca tacggtttcc ctggtatgcc aggtatgcct
ggtatg 11621117DNAArtificial SequencePrimer OVL-N2B-RV 21gcgtctgcag
tacgaattct attagccacc ggctggacca tcgtgaccgt gatgtccgtg 60gtgaccgggc
ttacccttgt ctcctggcat accaggcata cctggcatac cagggaa
11722599PRTArtificial SequenceRecombinant collagen-like polypeptide
N1N1P4 22Gly Pro Pro Gly Val Pro Gly Phe Ile Gly Phe Pro Gly Leu
Pro Gly1 5 10 15Trp Pro Gly Val Phe Gly Ile Pro Gly Tyr Pro Gly Tyr
Leu Gly Trp 20 25 30Pro Gly Trp Pro Gly Phe Pro Gly Ile Phe Gly Tyr
Pro Gly Tyr Pro 35 40 45Gly Trp Pro Gly Phe Pro Gly Trp Pro Gly Phe
Ile Gly Leu Pro Gly 50 55 60Tyr Leu Gly Pro Trp Gly Phe Val Gly Trp
Pro Gly Trp Leu Gly Tyr65 70 75 80Pro Gly Leu Phe Gly Leu Pro Gly
Tyr Pro Gly His Glu Gly Ile Pro 85 90 95Gly Asp His Gly Pro Ala Gly
Val Pro Gly Phe Ile Gly Phe Pro Gly 100 105 110Leu Pro Gly Trp Pro
Gly Val Phe Gly Ile Pro Gly Tyr Pro Gly Tyr 115 120 125Leu Gly Trp
Pro Gly Trp Pro Gly Phe Pro Gly Ile Phe Gly Tyr Pro 130 135 140Gly
Tyr Pro Gly Trp Pro Gly Phe Pro Gly Trp Pro Gly Phe Ile Gly145 150
155 160Leu Pro Gly Tyr Leu Gly Pro Trp Gly Phe Val Gly Trp Pro Gly
Trp 165 170 175Leu Gly Tyr Pro Gly Leu Phe Gly Leu Pro Gly Tyr Pro
Gly His Glu 180 185 190Gly Ile Pro Gly Asp His Gly Pro Ala Gly Glu
Pro Gly Asn Pro Gly 195 200 205Ser Pro Gly Asn Gln Gly Gln Pro Gly
Asn Lys Gly Ser Pro Gly Asn 210 215 220Pro Gly Gln Pro Gly Asn Glu
Gly Gln Pro Gly Gln Pro Gly Gln Asn225 230 235 240Gly Gln Pro Gly
Glu Pro Gly Ser Asn Gly Pro Gln Gly Ser Gln Gly 245 250 255Asn Pro
Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser Gln Gly Ser 260 265
270Pro Gly Asn Gln Gly Ser Pro Gly Gln Pro Gly Asn Pro Gly Gln Pro
275 280 285Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly Pro Ala Gly Glu
Pro Gly 290 295 300Asn Pro Gly Ser Pro Gly Asn Gln Gly Gln Pro Gly
Asn Lys Gly Ser305 310 315 320Pro Gly Asn Pro Gly Gln Pro Gly Asn
Glu Gly Gln Pro Gly Gln Pro 325 330 335Gly Gln Asn Gly Gln Pro Gly
Glu Pro Gly Ser Asn Gly Pro Gln Gly 340 345 350Ser Gln Gly Asn Pro
Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser 355 360 365Gln Gly Ser
Pro Gly Asn Gln Gly Ser Pro Gly Gln Pro Gly Asn Pro 370 375 380Gly
Gln Pro Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly Pro Ala Gly385 390
395 400Glu Pro Gly Asn Pro Gly Ser Pro Gly Asn Gln Gly Gln Pro Gly
Asn 405 410 415Lys Gly Ser Pro Gly Asn Pro Gly Gln Pro Gly Asn Glu
Gly Gln Pro 420 425 430Gly Gln Pro Gly Gln Asn Gly Gln Pro Gly Glu
Pro Gly Ser Asn Gly 435 440 445Pro Gln Gly Ser Gln Gly Asn Pro Gly
Lys Asn Gly Gln Pro Gly Ser 450 455 460Pro Gly Ser Gln Gly Ser Pro
Gly Asn Gln Gly Ser Pro Gly Gln Pro465 470 475 480Gly Asn Pro Gly
Gln Pro Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly 485 490 495Pro Ala
Gly Glu Pro Gly Asn Pro Gly Ser Pro Gly Asn Gln Gly Gln 500 505
510Pro Gly Asn Lys Gly Ser Pro Gly Asn Pro Gly Gln Pro Gly Asn Glu
515 520 525Gly Gln Pro Gly Gln Pro Gly Gln Asn Gly Gln Pro Gly Glu
Pro Gly 530 535 540Ser Asn Gly Pro Gln Gly Ser Gln Gly Asn Pro Gly
Lys Asn Gly Gln545 550 555 560Pro Gly Ser Pro Gly Ser Gln Gly Ser
Pro Gly Asn Gln Gly Ser Pro 565 570 575Gly Gln Pro Gly Asn Pro Gly
Gln Pro Gly Glu Gln Gly Lys Pro Gly 580 585 590Asn Gln Gly Pro Ala
Gly Gly 59523599PRTArtificial SequenceRecombinant collagen-like
polypeptide N1N2P4 23Gly Pro Pro Gly Val Pro Gly Phe Ile Gly Phe
Pro Gly Leu Pro Gly1 5 10 15Trp Pro Gly Val Phe Gly Ile Pro Gly Tyr
Pro Gly Tyr Leu Gly Trp 20 25 30Pro Gly Trp Pro Gly Phe Pro Gly Ile
Phe Gly Tyr Pro Gly Tyr Pro 35 40 45Gly Trp Pro Gly Phe Pro Gly Trp
Pro Gly Phe Ile Gly Leu Pro Gly 50 55 60Tyr Leu Gly Pro Trp Gly Phe
Val Gly Trp Pro Gly Trp Leu Gly Tyr65 70 75 80Pro Gly Leu Phe Gly
Leu Pro Gly Tyr Pro Gly His Glu Gly Ile Pro 85 90 95Gly Asp His Gly
Pro Ala Gly Val Pro Gly Phe Ile Gly Phe Pro Gly 100 105 110Leu Pro
Gly Trp Pro Gly Val Phe Gly Ile Pro Gly Tyr Pro Gly Tyr 115 120
125Leu Gly Trp Pro Gly Trp Pro Gly Trp Pro Gly Pro Phe Gly Trp Leu
130 135 140Gly Pro Phe Gly Tyr Pro Gly Ile Tyr Gly Trp Pro Gly Phe
Leu Gly145 150 155 160Tyr Pro Gly Ile Phe Gly Pro Trp Gly Pro Tyr
Gly Phe Pro Gly Met 165 170 175Pro Gly Met Pro Gly Met Pro Gly Asp
Lys Gly Lys Pro Gly His His 180 185 190Gly His His Gly His Asp Gly
Pro Ala Gly Glu Pro Gly Asn Pro Gly 195 200 205Ser Pro Gly Asn Gln
Gly Gln Pro Gly Asn Lys Gly Ser Pro Gly Asn 210 215 220Pro Gly Gln
Pro Gly Asn Glu Gly Gln Pro Gly Gln Pro Gly Gln Asn225 230 235
240Gly Gln Pro Gly Glu Pro Gly Ser Asn Gly Pro Gln Gly Ser Gln Gly
245 250 255Asn Pro Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser Gln
Gly Ser 260 265 270Pro Gly Asn Gln Gly Ser Pro Gly Gln Pro Gly Asn
Pro Gly Gln Pro 275 280 285Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly
Pro Ala Gly Glu Pro Gly 290 295 300Asn Pro Gly Ser Pro Gly Asn Gln
Gly Gln Pro Gly Asn Lys Gly Ser305 310 315 320Pro Gly Asn Pro Gly
Gln Pro Gly Asn Glu Gly Gln Pro Gly Gln Pro 325 330 335Gly Gln Asn
Gly Gln Pro Gly Glu Pro Gly Ser Asn Gly Pro Gln Gly 340 345 350Ser
Gln Gly Asn Pro Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser 355 360
365Gln Gly Ser Pro Gly Asn Gln Gly Ser Pro Gly Gln Pro Gly Asn Pro
370 375 380Gly Gln Pro Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly Pro
Ala Gly385 390 395 400Glu Pro Gly Asn Pro Gly Ser Pro Gly Asn Gln
Gly Gln Pro Gly Asn 405 410 415Lys Gly Ser Pro Gly Asn Pro Gly Gln
Pro Gly Asn Glu Gly Gln Pro 420 425 430Gly Gln Pro Gly Gln Asn Gly
Gln Pro Gly Glu Pro Gly Ser Asn Gly 435 440 445Pro Gln Gly Ser Gln
Gly Asn Pro Gly Lys Asn Gly Gln Pro Gly Ser 450 455 460Pro Gly Ser
Gln Gly Ser Pro Gly Asn Gln Gly Ser Pro Gly Gln Pro465 470 475
480Gly Asn Pro Gly Gln Pro Gly Glu Gln Gly Lys Pro Gly Asn Gln Gly
485 490 495Pro Ala Gly Glu Pro Gly Asn Pro Gly Ser Pro Gly Asn Gln
Gly Gln 500 505 510Pro Gly Asn Lys Gly Ser Pro Gly Asn Pro Gly Gln
Pro Gly Asn Glu 515 520 525Gly Gln Pro Gly Gln Pro Gly Gln Asn Gly
Gln Pro Gly Glu Pro Gly 530 535 540Ser Asn Gly Pro Gln Gly Ser Gln
Gly Asn Pro Gly Lys Asn Gly Gln545 550 555 560Pro Gly Ser Pro Gly
Ser Gln Gly Ser Pro Gly Asn Gln Gly Ser Pro 565 570 575Gly Gln Pro
Gly Asn Pro Gly Gln Pro Gly Glu Gln Gly Lys Pro Gly 580 585 590Asn
Gln Gly Pro Ala Gly Gly 5952421DNAArtificial SequencePrimer
24gactggttcc aattgacaag c 212521DNAArtificial SequencePrimer
25gcaaatggca ttctgacatc c 21
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