U.S. patent application number 16/654232 was filed with the patent office on 2020-07-16 for scalable fermentation process.
The applicant listed for this patent is KUROS BIOSCIENCES AG. Invention is credited to Marcel EMMERLING, Frank HENNECKE, Holger PFRUNDER, Martin RHIEL, Philipp STEINER.
Application Number | 20200223891 16/654232 |
Document ID | / |
Family ID | 37434299 |
Filed Date | 2020-07-16 |
![](/patent/app/20200223891/US20200223891A1-20200716-D00001.png)
United States Patent
Application |
20200223891 |
Kind Code |
A1 |
EMMERLING; Marcel ; et
al. |
July 16, 2020 |
SCALABLE FERMENTATION PROCESS
Abstract
This invention provides a robust fermentation process for the
expression of a capsid protein of a bacteriophage which is forming
a VLP by self-assembly, wherein the process is scalable to a
commercial production scale and wherein the expression rate of the
capsid protein is controlled to obtain improved yield of soluble
capsid protein. This is achieved by combining the advantages of
fed-batch culture and of lactose induced expression systems with
specific process parameters providing improved repression of the
promoter during the growth phase and high plasmid retention
throughout the process.
Inventors: |
EMMERLING; Marcel;
(Schlieren, CH) ; HENNECKE; Frank; (Dietlikon,
CH) ; PFRUNDER; Holger; (Zurich, CH) ; RHIEL;
Martin; (Bonstetten, CH) ; STEINER; Philipp;
(Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KUROS BIOSCIENCES AG |
Schlieren |
|
CH |
|
|
Family ID: |
37434299 |
Appl. No.: |
16/654232 |
Filed: |
October 16, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15353884 |
Nov 17, 2016 |
|
|
|
16654232 |
|
|
|
|
14247097 |
Apr 7, 2014 |
9518095 |
|
|
15353884 |
|
|
|
|
13335008 |
Dec 22, 2011 |
|
|
|
14247097 |
|
|
|
|
11921023 |
Nov 26, 2007 |
|
|
|
PCT/EP2006/062628 |
May 24, 2006 |
|
|
|
13335008 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2795/10061
20130101; A61K 2039/5256 20130101; C12N 2795/00023 20130101; C12N
7/00 20130101; C12N 2795/18022 20130101; C12N 2795/10051 20130101;
C12N 2795/18052 20130101; C07K 14/005 20130101; A61K 2039/5258
20130101; C12N 2795/00051 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 26, 2005 |
EP |
05011416.4 |
Jul 21, 2005 |
EP |
05106729.6 |
Claims
1. A process for expression of a recombinant capsid protein of a
RNA bacteriophage being capable of forming a virus-like particle
(VLP) by self-assembly, wherein said RNA bacteriophage is RNA
bacteriophage Q.beta., and wherein said recombinant capsid protein
comprises SEQ ID NO:5, said process comprising: a.) introducing an
expression plasmid into a bacterial host, wherein said expression
plasmid comprises an expression construct, wherein said expression
construct comprises (i) a first nucleotide sequence encoding said
recombinant capsid protein, or mutant or fragment thereof, and (ii)
a promoter being inducible by lactose; b.) initiating a growth
phase by cultivating said bacterial host in a medium comprising a
major carbon source; wherein said cultivating initiates a batch
phase during said growth phase, wherein said cultivating is
performed in batch culture and under conditions under which said
promoter is repressed by lacI, wherein said lacI is overexpressed
by said bacterial host, and wherein no feeding of said batch
culture is performed during said cultivating; c.) ending said batch
phase and initiating a feed phase during said growth phase by
feeding said batch culture with said major carbon source; wherein
said feeding of said batch culture is performed with a flow rate,
wherein said flow rate increases with an exponential coefficient
.mu., and d.) ending said growth phase and initiating a production
phase by inducing said promoter with an inducer, wherein said
feeding of said batch culture with said major carbon source is
continued; and wherein during said steps (b.), (c.) and (d.) no
removal of medium, except for analytical purposes, takes place
leading to an increased density of said bacterial host in said
medium.
2-5. (canceled)
6. The process of claim 1, wherein said expression construct
comprises a first stop codon and a second stop codon, wherein said
first stop codon is located directly 3' of said first nucleotide
sequence and wherein said second stop codon is located directly 3'
of said first stop codon, and wherein at least one of said first or
second stop codon is TAA.
7. The process of claim 1, wherein said expression construct
further comprises a second nucleotide sequence, wherein said first
nucleotide sequence encodes Q.beta. coat protein (CP), and wherein
said second nucleotide sequence encodes Q.beta. A1 protein and
wherein said first and said second nucleotide sequence are
separated by exactly one sequence stretch comprising at least one
TAA stop codon.
8. The process of claim 1, wherein said expression construct
comprises SEQ ID NO:6.
9. The process of claim 1, wherein said expression plasmid
comprises SEQ ID NO:1.
10. (canceled)
11. The process of claim 1, wherein said promoter is selected from
the group consisting of a.) tac promoter; b.) trc promoter; c.) tic
promoter; d.) lac promoter; e.) lacUV5 promoter; f.) P.sub.syn
promoter; g.) lpp.sup.a promoter; h.) lpp-lac promoter; i.) T7-lac
promoter; j.) T3-lac promoter; k.) T5-lac promoter; and l.) a
promoter having at least 50% sequence homology to SEQ ID NO:2.
12. The process of claim 1, wherein said promoter comprises SEQ ID
NO:2.
13. The process of claim 1, wherein said major carbon source is
glycerol.
14. The process of claim 1, wherein said exponential coefficient
.mu. is below .mu..sub.max.
15. The process of claim 1, wherein said inducing of said promoter
is performed by co-feeding said batch culture with said inducer and
said major carbon source at a constant flow rate.
16. The process of claim 1, wherein said inducing of said promoter
is performed by co-feeding said batch culture with said inducer and
said major carbon source at an increasing flow rate.
17. The process of claim 15, wherein said inducer is lactose and
wherein said lactose and said major carbon source are co-fed to
said batch culture in a ratio of about 2:1 to 1:4 (w/w).
18. The process of claim 1, wherein said inducer is IPTG and
wherein the concentration of said IPTG in said medium is 0.001 to 5
mM.
19. (canceled)
20. The process of claim 1, wherein said lacI is overexpressed by
said bacterial host, wherein said overexpression is caused by
lacI.sup.q or lacQ1.
21. (canceled)
22. (canceled)
23. The process of claim 1, wherein said inducer is lactose and
wherein said bacterial host comprises .beta.-galactosidase
activity.
24. The process of claim 1, wherein said cultivating and said
feeding of said batch culture and said inducing of said promoter is
performed at a temperature which is below the optimal growth
temperature of said bacterial host.
25. The process of claim 1 wherein: a.) said major carbon source is
glycerol; b.) said inducer is lactose; c.) and said lactose and
said major carbon source are co-fed to said batch culture in a
ratio of 2:1 to 1:4 (w/w); d.) said bacterial host is E. coli
RB791; and e.) said cultivating and feeding of said batch culture
and said inducing of said promoter is performed at a temperature of
about 30.degree. C.
26. (canceled)
27. The process of claim 1, wherein throughout steps b.) to d.) of
said process oxygen is supplied to said bacterial host, wherein
said oxygen supply is effected such that the partial pressure of
oxygen in the medium (pO.sub.2) is at least about 40%.
28. The process of claim 16, wherein said inducer is lactose and
wherein said lactose and said major carbon source are co-fed to
said batch culture in a ratio of about 2:1 to 1:4 (w/w).
29. The process of claim 1, wherein said inducer is lactose.
30. The process of claim 20, wherein said overexpression is caused
by lacI.sup.q.
31. The process of claim 29, wherein said lactose and said major
carbon source are co-fed to said batch culture in a ratio of 1:1 to
1:3 (w/w).
32. The process of claim 29, wherein said lactose and said major
carbon source are co-fed to said batch culture in a ratio of 1:3
(w/w).
Description
FIELD OF THE INVENTION
[0001] This invention is related to the field of protein expression
and fermentation technology. A process for the efficient expression
of recombinant bacteriophage capsid protein in a bacterial host is
described. The process leads to high yield of recombinant capsid
protein which is capable of forming a virus-like particle (VLP) by
self-assembly. Furthermore, the process is scalable from laboratory
scale to fermenter volumes larger than 50 litres.
BACKGROUND OF THE INVENTION
[0002] Recent vaccination strategies make use of viruses or
virus-like-particles (VLPs) to enhance the immune response towards
antigens. For example, WO02/056905 demonstrates the utility of VLPs
as a carrier to present antigens linked thereto in a highly ordered
repetitive array. Such antigen arrays can cause a strong immune
response, in particular antibody responses, against the linked
antigen and are even capable of breaking the immune system's
inherent tolerance towards self antigens. Such antigen arrays are
therefore useful in the production of vaccines for the treatment of
infectious diseases and allergies as well as for the efficient
induction of self-specific immune responses, e.g. for the treatment
of cancer, rheumatoid arthritis and various other diseases.
[0003] As indicated in WO02/056905 capsid proteins of
bacteriophages are particularly suited as antigen carrier. They
have been shown to efficiently self-assemble into VLPs upon
expression in a bacterial host (Kastelein et al. 1983, Gene
23:245-254; Kozlovskaya et al. 1986, Dokl. Akad. Nauk SSSR
287:452-455). Moreover, capsid proteins of bacteriophages such as
derived from fr (Pushko et al. 1993, Protein Engineering
6(8)883-891), Q.beta. (Kozlovska et al. 1993, Gene 137:133-137;
Ciliens et al. 2000, FEBS Letters 24171:1-4; Vasiljeva et al 1998,
FEBS Letters 431:7-11) and MS-2 (WO92/13081; Mastico et al. 1993,
Journal of General Virology 74:541-548; Heal et al. 2000, Vaccine
18:251-258) have been produced in bacterial hosts using inducible
promoters such as the trp promoter or a trp-T7 fusion (in the case
of fr and Qb) or the tac promoter using IPTG as inducer substance
(in the case of MS-2). The use of inducible promoters is
beneficial, to avoid possible toxic effects of the recombinant
capsid protein and the metabolic burden of protein expression which
both might reduce the growth of the bacterial expression host and,
ultimately, the yield of expressed protein.
[0004] However, the expression systems used so far for the
expression of capsid proteins of bacteriophages have been applied
in small scale fermentations, i.e. in laboratory scale and small
batch cultures with volumes of typically clearly below 1 litre. An
scale up of these systems comprising volumes of 50 litre and more
is expected to diminish in a great extent the respective capsid
protein yield due to increased promoter leakage and/or lowered
plasmid retention.
[0005] A further problem associated with commercially desired
high-level expression and rapid accumulation of recombinant capsid
proteins of bacteriophages is the formation of incorrectly folded
protein species and the formation of so called inclusion bodies,
i.e. protein aggregates, which are insoluble and which may hamper
further downstream processes. Thus, for bacteriophage MS-2 coat
protein the formation of protein aggregates and of protein species
which lost their ability to self-assemble to VLPs have been
reported when the protein was expressed under the control of the
strong T7 promoter after IPTG induction using the pET expression
system (Peabody & Al-Bitar 2001, Nucleic Acid Research
29(22):e113).
[0006] High expression rates of the recombinant capsid protein may
therefore have a negative impact on the yield of correctly
assembled VLPs. The production of VLP-based vaccines in a
commercial scale requires, therefore, the establishment of an
efficient, and in particular scalable fermentation process for the
expression of recombinant capsid protein of bacteriophages leading
to a product of constant quality and purity having the capability
of self-assembling into VLPs, whereby the formation of insoluble
fractions of the capsid protein is minimised or avoided.
[0007] Therefore, it is an object of the present invention to
provide a process for expression of a recombinant capsid protein of
a bacteriophage which avoids or minimizes the disadvantage or
disadvantages of the prior art processes, and in particular, which
is scalable to a commercial scale and still leading to a product of
constant quality and purity and the capability of self-assemblance
to VLPs, and wherein the formation of insoluble fraction of the
capsid protein is minimised or avoided.
SUMMARY OF THE INVENTION
[0008] The invention relates to a process for expression of a
recombinant capsid protein of a bacteriophage, or a mutant or
fragment thereof being capable of forming a VLP by self-assembly,
said process comprising the steps of:.a) introducing an expression
plasmid into a bacterial host, wherein said expression plasmid
comprises an expression construct, wherein said expression
construct comprises (i) a first nucleotide sequence encoding said
recombinant capsid protein, or mutant or fragment thereof, and (ii)
a promoter being inducible by lactose; b.) cultivating said
bacterial host in a medium comprising a major carbon source;
wherein said cultivating is performed in batch culture and under
conditions under which said promoter is repressed by lad, wherein
said lad is overexpressed by said bacterial host; c.) feeding said
batch culture with said major carbon source; and d.) inducing said
promoter with an inducer, wherein preferably said feeding of said
batch culture with said major carbon source is continued.
[0009] This invention provides a robust fermentation process for
the expression of a capsid protein of a bacteriophage which is
forming a VLP by self-assembly, wherein the process is scalable to
a commercial production scale and wherein the expression rate of
the capsid protein leads to improved yield of soluble capsid
protein. This is, in particular, achieved by improved repression of
the promoter during the growth phase and high plasmid retention
throughout the process. The expression system further avoids
formation of insoluble protein aggregates by limiting the maximum
expression rate occurring during the production phase.
[0010] In a preferred embodiment said bacteriophage is a RNA
bacteriophage. More preferably, said RNA bacteriophage is selected
from the group consisting of: a.) bacteriophage Q.beta.; b.)
bacteriophage AP205; c.) bacteriophage fr; d.) bacteriophage GA;
e.) bacteriophage SP; f.) bacteriophage MS2; g.) bacteriophage M11;
h.) bacteriophage MX1; i.) bacteriophage NL95; j.) bacteriophage
f2; k.) bacteriophage PP7 and 1.) bacteriophage R17. Preferably,
said RNA bacteriophage is Q.beta.. More preferably said recombinant
capsid protein comprises or alternatively consists of an amino acid
sequence selected from the group consisting of SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
Still more preferably said recombinant capsid protein comprises SEQ
ID NO:5, most preferably said recombinant capsid protein consists
of SEQ ID NO:5.
[0011] In a further preferred embodiment said recombinant capsid
protein comprises or alternatively consists of an amino acid
sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:13, and SEQ ID NO:14. More preferably said recombinant capsid
protein comprises SEQ ID NO:12, most preferably said recombinant
capsid protein consists of SEQ ID NO:12.
[0012] In another embodiment of the present invention, said
expression construct comprises a first stop codon, and wherein said
first stop codon is TAA, and wherein preferably said TAA is located
directly 3' of said first nucleotide sequence.
[0013] In a further embodiment said expression construct comprises
a first stop codon and a second stop codon, wherein said first stop
codon is located directly 3' of said first nucleotide sequence and
wherein said second stop codon is located directly 3' of said first
stop codon, and wherein at least one of said first or second stop
codon is TAA.
[0014] In a further embodiment said expression construct comprises
a first nucleotide sequence and a second nucleotide sequence,
wherein said first nucleotide sequence is encoding a recombinant
capsid protein, preferably Q.beta. CP, or a mutant or fragment
thereof, and wherein said second nucleotide sequence is encoding
any other protein, preferably the Q.beta. A1 protein or a mutant or
fragment thereof, and wherein said first and said second nucleotide
sequence are separated by exactly one sequence stretch comprising
at least one TAA stop codon. In a preferred embodiment said
expression construct comprises or alternatively consists of the
nucleotide sequence of SEQ ID NO:6.
[0015] In a further embodiment said expression plasmid comprises
or, more preferably, consists of the nucleotide sequence of SEQ ID
NO:1.
[0016] In one embodiment of the invention said promoter is selected
from the group consisting of the a.) tac promoter; b.) trc
promoter; c.) tic promoter; d.) lac promoter; e.) lacUV5 promoter;
f.) P.sub.syn promoter; g.) 1pp.sup.a promoter; h.) 1pp-lac
romoter; i.) T7-lac promoter; j.) T3-lac promoter; k.) T5-lac
promoter; and 1.) a promoter having at least 50% sequence homology
to SEQ ID NO:2.
[0017] In a preferred embodiment said promoter has at least 50%,
60%, 70%, 80, 90, or 95%, preferably 98 to 100%, most preferably
99% sequence homology to SEQ ID NO:2. In a further preferred
embodiment said promoter is selected from the group consisting of
tic promoter, trc promoter and tac promoter. Even more preferably
said promoter is the tac promoter. Most preferably said promoter
comprises or alternatively consists of the nucleotide sequence of
SEQ ID NO:2.
[0018] In one embodiment said major carbon source is glucose or
glycerol, preferably glycerol.
[0019] In one embodiment said feeding of said batch culture is
performed with a flow rate, wherein said flow rate increases with
an exponential coefficient .mu., and wherein preferably said
exponential coefficient .mu. is below .mu..sub.max.
[0020] In a further embodiment said inducing of said promoter is
performed by co-feeding said batch culture with said inducer,
preferably lactose and said major carbon source, preferably
glycerol, at a constant flow rate.
[0021] In a further embodiment said inducing of said promoter is
performed by co-feeding said batch culture with said inducer,
preferably lactose and said major carbon source, preferably
glycerol, at an increasing flow rate.
[0022] In a further embodiment said inducer is lactose, wherein
preferably said lactose and said major carbon source are co-fed to
said batch culture in a ratio of about 2:1 to 1:4 (w/w).
[0023] In a further embodiment said inducer is IPTG wherein
preferably the concentration of said IPTG said medium is 0.001 to 5
mM, preferably 0.001 to 1 mM, more preferably 0.005 to 1 mM, still
more preferably 0.005 to 0.5 mM. In a very preferred embodiment
said concentration of IPTG is about 0.01 mM, most preferably 0.01
mM.
[0024] In one embodiment said lad is overexpressed by said
bacterial host, wherein said overexpression is caused by lacI.sup.q
or lacQ1, preferably by lacI.sup.q. In one embodiment said
bacterial host comprises said lacI.sup.q gene or said lacQ1 gene,
preferably said lacI.sup.q gene on its chromosome. In a further
prefered embodiment said bacterial host comprises said lacI.sup.q
gene or said lacQ1 gene, preferably said lacI.sup.q gene on a
plasmid, preferably on a high copy number plasmid. In a further
prefered embodiment said bacterial host comprises said lacI.sup.q
gene or said lacQ1 gene, preferably said lacI.sup.q gene on said
expression plasmid.
[0025] In one embodiment said bacterial host is selected from the
group consisting of the strains E. coli RB791, E. coli DH2O and E.
coli Y1088. Preferably said bacterial host is E. coli RB791.
[0026] In one embodiment said bacterial host comprises
.beta.-galactosidase activity.
[0027] In one embodiment said cultivating and said feeding of said
batch culture and said inducing of said promoter is performed at a
temperature which is below the optimal growth temperature of said
bacterial host. Preferably said temperature is between 23.degree.
C. and 35.degree. C., more preferably between 25 and 33.degree. C.,
even more preferably between 27 and 32.degree. C., still more
preferably between 28 and 31.degree. C. Even more preferably said
temperature is about 30.degree. C., most preferably said
temperature is 30.degree. C.
[0028] In one embodiment said cultivating and said feeding of said
batch culture is performed at a temperature which is below the
optimal growth temperature of said bacterial host, wherein
preferably said temperature is between 23.degree. C. and 35.degree.
C., more preferably between 25 and 33.degree. C., even more
preferably between 27 and 32.degree. C., still more preferably
between 28 and 31.degree. C., even more preferably said temperature
is about 30.degree. C., most preferably said temperature is
30.degree. C., and said inducing of said promoter is performed at
the optimal growth temperature of the bacterial host, preferably at
about 37.degree. C.
[0029] In one embodiment said cultivating and said feeding of said
batch culture and said inducing of said promoter is performed in
the absence of an antibiotic.
[0030] In a specific embodiment said expression plasmid comprises
or alternatively consists of the nucleotide sequence of SEQ ID
NO:1, said major carbon source is glycerol, said feeding of said
batch culture is performed with a flow rate, wherein said flow rate
increases with an exponential coefficient .mu., and wherein said
exponential coefficient .mu. is below .mu..sub.max, said inducing
of said promoter by co-feeding said batch culture is performed with
a constant flow rate, wherein lactose and glycerol are co-fed to
the batch culture in a ratio of about 2:1 to about 1:4 (w/w),
preferably about 1:1 to about 1:4 (w/w), most preferably about 1:3
(w/w), and wherein said cultivating and feeding of said batch
culture and said inducing of said promoter is performed at a
temperature between 27 and 32.degree. C., preferably about
30.degree. C., most preferably 30.degree. C.
[0031] In a further specific embodiment said expression plasmid
comprises or alternatively consists of the nucleotide sequence of
SEQ ID NO:30, said major carbon source is glycerol, said feeding of
said batch culture is performed with a flow rate, wherein said flow
rate increases with an exponential coefficient .mu., and wherein
said exponential coefficient .mu. is below .mu..sub.max, said
inducing of said promoter by co-feeding said batch culture is
performed with a constant flow rate, wherein lactose and said major
carbon source are co-fed to the batch culture in a ratio of about
2:1 to about 1:4 (w/w), preferably about 1:1 to about 1:4 (w/w),
most preferably about 1:3 (w/w), and wherein said cultivating and
feeding of said batch culture and said inducing of said promoter is
performed at a temperature between 27 and 32.degree. C., preferably
about 30.degree. C., most preferably 30.degree. C.
DESCRIPTION OF THE FIGURES
[0032] FIG. 1: Fermentation profile with pTac-nSD-Qb-mut (SEQ ID
NO:1) in RB791 in 21 culture. Co-feeding during production phase
was performed with medium containing 20% glycerol and 20% lactose.
Shown are glycerol concentration [g/l] (circles); lactose
concentration [g/l] (triangles); .beta.-Gal activity [U/ml*OD=1]
(squares) and OD600 (diamonds) plotted against the process time
[h].
DETAILED DESCRIPTION OF THE INVENTION
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0034] "about": within the meaning of the present application the
expression about shall have the meaning of +/-10%. For example
about 100 shall mean 90 to 110.
[0035] "promoter which is inducible by lactose" as used herein
refers to a promoter which comprises regulatory elements of the lac
operon. Such promoters are repressed by lad and can be induced by
lactose or the synthetic inducer IPTG. The skilled person is aware
that induction of a promoter by lactose requires
.beta.-galactosidase activity in the bacterial host.
[0036] "located directly 3'": a nucleotide sequence N2 which is
located directly 3' of another nucleotide sequence N1 refers to a
continuous sequence having the conformation 5'-N1-N2-3' wherein N1
and N2 are directly connected and not separated by additional
sequence elements.
[0037] "sequence stretch": as used herein the term "sequence
stretch" refers to a continuous nucleotide sequence which consists
of less than 50, preferably less than 20, more preferably less than
10, even more preferably less than 5 nucleotides. In a further
preferred embodiment the sequence stretch comprises or
alternatively consists of at least one, preferably one, TAA stop
codon. In another embodiment the sequence stretch comprises or
alternatively consists of at least one, preferably one, TAA and at
least one, preferably one, TGA stop codon. In further preferred
embodiment the sequence stretch comprises or alternatively consists
of SEQ ID NO:32.
[0038] "bacterial host": as used herein the term "bacterial host"
refers to a bacterial organism which is hosting or capable of
hosting an expression plasmid of the invention, wherein "hosting"
involves the replication of the expression plasmid and maintenance
of the expression plasmid during cell division.
[0039] "culture": in the context of the instant invention a
"culture" comprises a bacterial host in a medium ("bacterial
culture"), wherein typically said medium is supporting the growth
of said bacterial host.
[0040] "batch culture" as used herein relates to a culture, i.e. a
bacterial host in a medium, wherein said culture constitutes a
closed system, i.e. typically and preferably no addition or removal
of medium takes place during the cultivation time. Therefore, in
contrast to a continuous culture, typically and preferably the
density of the bacterial host in the batch culture continuously
increases with progressing cultivation time. Batch culture does not
exclude the addition of compounds required for the control of the
process, such as, for example, inducer, oxygen, and alkali or acid
to control the pH.
[0041] "fed batch culture": as used herein is a culture which is
supplied with additional medium comprising a substrate, preferably
the major carbon source of the bacterial host (feed or co-feed
medium). In the context of the application this process is referred
to by the terms "feeding said batch culture" (medium comprises the
major carbon source) and "co-feeding said batch culture" (medium
comprises the major carbon source and the inducer, preferably
lactose). Typically and preferably, no removal of medium except for
analytical purposes takes place during cultivation time of a fed
batch culture.
[0042] "Preculture": a culture, preferably a batch culture, which
is used to produce the inoculum for a culture of a larger volume,
e.g. the culture in which the recombinant capsid protein is
produced (production culture). A preculture can be performed in two
or more steps, wherein a second preculture is inoculated with a
first preculture etc. to produce a sufficiently large inoculum for
the production culture. The first and/or subsequent precultures may
comprise an antibiotic to improve plasmid stability.
[0043] "substrate": as used herein refers to a compound in the
culture medium which contributes to the carbon and energy supply of
the bacterial host. The terms "substrate" therefore encompasses any
compound contained in the medium contributing to the carbon supply
of the bacterial host. Typical substrates for bacteria are sugar,
starch, glycerol, acetate and any other organic compound which can
be metabolized by bacteria. Therefore, the term "substrate"
includes the major carbon source but also, for example,
lactose.
[0044] "Major carbon source" as used herein refers to the compound
in the culture medium which contributes most to the carbon and
energy supply of the bacterial host during the growth phase. The
major carbon source thus is the major substrate of the bacterial
host. The major carbon source is typically a sugar such as sucrose
or glucose, or glycerol, and preferably glucose or glycerol. Though
lactose could in principal act as a major carbon source for a
bacterial host, in the context of the instant invention the term
"major carbon source" typically and preferably does not include
lactose.
[0045] Phases of the process of the invention: The process of the
invention is characterised by different phases which refer to
different physiological conditions of the bacterial host with
respect to its growth and the repression/induction status of the
expression construct.
[0046] "Growth phase": The growth phase is initiated by said
cultivating said bacterial host in a medium. The growth phase is
preferably characterized by conditions under which the promoter
driving the expression of the recombinant capsid protein is
repressed and the growth phase is terminated with said inducing
said promoter with an inducer. The growth phase can be further
divided in a "batch phase" and a "feed phase". Said batch phase is
initiated by said cultivating said bacterial host in a medium. The
batch phase comprised a "lag phase" during which the bacterial host
is not yet growing or growing with a non-exponential rate,
typically and preferably a linear rate. The growth phase further
comprises an "exponential growth phase" which directly follows the
lag phase. No feeding of said culture takes place during the batch
phase, thus the exponential growth phase is terminated by the
consumption of the substrate by the bacterial host. The growth
phase further comprises a "feed phase" which is directly following
the batch phase and which is initiated by said feeding of said
batch culture with said major carbon source. The feed phase is
characterised by a growth rate of the bacterial host which is
directly dependent on the flow rate of the feed medium containing
the major carbon source.
[0047] "production phase": The growth phase is followed by the
production phase which is initiated by said inducing said promoter
with an inducer, wherein typically and preferably said feeding of
said batch culture with said major carbon source is continued.
[0048] "Conditions under which the promoter is repressed": it is to
be understood that the repression of a promoter is an equilibrium
of formation and dissociation of the repressor-operator complex and
that even stringently repressed promoters may show a certain
expression rate also in the absence of their inducer. Therefore, as
used within the application the term "conditions under which the
promoter is repressed" relates to conditions, wherein at the end of
the growth phase, i.e. directly before the addition of inducer to
the culture, the recombinant capsid protein is expressed to a level
which does not exceed a concentration in the medium of 200 mg/l,
preferably 150 mg/l, more preferably 100 mg/l, as determined by the
HLPC method of Example 17. Most preferably, the concentration of
the recombinant protein is below the detection level of said
method.
[0049] "Inducer": within the meaning of the in invention the term
"inducer" relates to any substance which directly or indirectly
interacts with an inducible promoter and thereby facilitates
expression from said promoter; for example, inducers of "a promoter
inducible by lactose", such as the lac or tac promoter, are IPTG,
lactose and allolactose.
[0050] "Coat protein"/"capsid protein": The term "coat protein" and
the interchangeably used term "capsid protein" within this
application, refers to a viral protein, preferably a subunit of a
natural capsid of a virus, preferably of a RNA bacteriophage, which
is capable of being incorporated into a virus capsid or a VLP. For
example, the specific gene product of the coat protein gene of RNA
bacteriophage Q.beta. is referred to as "Q.beta. CP", whereas the
"coat proteins" or "capsid proteins" of bacteriophage Q.beta.
comprise the "Q.beta. CP" as well as the Al protein.
[0051] "Recombinant capsid protein": A capsid protein which is
synthesised by a recombinant host cell. "Polypeptide": As used
herein the term "polypeptide" refers to a polymer composed of amino
acid residues, generally natural amino acid residues, linked
together through peptide bonds. Although a polypeptide may not
necessarily be limited in size, the term polypeptide is often used
in conjunction with peptide of a size of about ten to about 50
amino acids.
[0052] "Protein": As used herein, the term protein refers to a
polypeptide generally of a size of above 20, more particularly of
above 50 amino acid residues. Proteins generally have a defined
three dimensional structure although they do not necessarily need
to, and are often referred to as folded, in opposition to peptides
and polypeptides which often do not possess a defined
three-dimensional structure, but rather can adopt a large number of
different conformations, and are referred to as unfolded.
[0053] "Recombinant host cell": As used herein, the term
"recombinant host cell" refers to a host cell into which one ore
more nucleic acid molecules of the invention have been
introduced.
[0054] "Recombinant VLP": The term "recombinant VLP", as used
herein, refers to a VLP that is obtained by a process which
comprises at least one step of recombinant DNA technology. The term
"VLP recombinantly produced", as used herein, refers to a VLP that
is obtained by a process which comprises at least one step of
recombinant DNA technology. Thus, the terms "recombinant VLP" and
"VLP recombinantly produced" are interchangeably used herein and
should have the identical meaning.
[0055] "RNA-bacteriophage": As used herein, the term
"RNA-bacteriophage" refers to RNA viruses infecting bacteria,
preferably to single-stranded positive-sense RNA viruses infecting
bacteria.
[0056] "Virus-like particle (VLP)": as used herein, the term
"virus-like particle" refers to a structure resembling a virus
particle or it refers to a non-replicative or non-infectious,
preferably a non-replicative and non-infectious virus particle, or
it refers to a non-replicative or non-infectious, preferably a
non-replicative and non-infectious structure resembling a virus
particle, preferably a capsid of a virus. The term
"non-replicative", as used herein, refers to being incapable of
replicating the genome comprised by the VLP. The term
"non-infectious", as used herein, refers to being incapable of
entering the host cell. Preferably a virus-like particle in
accordance with the invention is non-replicative and/or
non-infectious since it lacks all or part of the viral genome or
genome function. Typically a virus-like particle lacks all or part
of the replicative and infectious components of the viral genome. A
virus-like particle in accordance with the invention may contain
nucleic acid distinct from their genome. A typical and preferred
embodiment of a virus-like particle in accordance with the present
invention is a viral capsid such as the viral capsid of the
corresponding virus, bacteriophage, preferably RNA-phage. The terms
"viral capsid" or "capsid", refer to a macromolecular assembly
composed of viral protein subunits. Typically, there are 60, 120,
180, 240, 300, 360 and more than 360 viral protein subunits.
Typically and preferably, the interactions of these subunits lead
to the formation of viral capsid or viral-capsid like structure
with an inherent repetitive organization, wherein said structure
is, typically, spherical or tubular. For example, the capsids of
RNA bacteriophages or HBcAgs have a spherical form of icosahedral
symmetry.
[0057] "Virus-like particle of a RNA bacteriophage": As used
herein, the term "virus-like particle of a RNA bacteriophage"
refers to a virus-like particle comprising, or preferably
consisting essentially of or consisting of coat proteins, mutants
or fragments thereof, of a RNA bacteriophage. In addition,
virus-like particle of a RNA bacteriophage resembling the structure
of a RNA bacteriophage, being non replicative and/or
non-infectious, and lacking at least the gene or genes encoding for
the replication machinery of the RNA bacteriophage, and typically
also lacking the gene or genes encoding the protein or proteins
responsible for viral attachment to or entry into the host.
Preferred VLPs derived from RNA bacteriophages exhibit icosahedral
symmetry and consist of 180 subunits. A preferred method to render
a virus-like particle of a RNA bacteriophage non replicative and/or
non-infectious is by genetic manipulation.
[0058] one, a, or an: When the terms "one," "a," or "an" are used
in this disclosure, they mean "at least one" or "one or more,"
unless otherwise indicated.
[0059] "Sequence identity": The amino acid sequence identity of
polypeptides can be determined conventionally using known computer
programs such as the Bestfit program. When using Bestfit or any
other sequence alignment program, preferably using Bestfit, to
determine whether a particular sequence is, for instance, 95%
identical to a reference amino acid sequence, the parameters are
set such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed. This aforementioned method in
determining the percentage of identity between polypeptides is
applicable to all proteins, polypeptides or a fragment thereof
disclosed in this invention.
[0060] "Sequence homology": The homology of nucleotide sequences
can for example be determined by the program blastn which is an
implementation of the BLAST algorithm, preferably using the default
settings of the software.
[0061] "Fragment of a protein", in particular fragment of a
recombinant protein or recombinant coat protein, as used herein, is
defined as a polypeptide, which is of at least 70%, preferably at
least 80%, more preferably at least 90%, even more preferably at
least 95% the length of the wild-type recombinant protein, or coat
protein, respectively and which preferably retains the capability
of forming VLP Preferably the fragment is obtained by at least one
internal deletion, at least one truncation or at least one
combination thereof. Further preferably the fragment is obtained by
at most 10, at most 9, at most 8, at most 7, at most 6, at most 5,
at most 4, at most 3 or at most 2 internal deletions; by at most
10, at most 9, at most 8, at most 7, at most 6, at most 5, at most
4, at most 3 or at most 2 truncations; or by at most 3, preferably
at most 2, most preferably by exactly one combination thereof. Most
preferably the fragment is obtained by exactly one internal
deletion, exactly one truncation or by a combination thereof.
[0062] The term "fragment of a recombinant protein" or "fragment of
a coat protein" shall further encompass polypeptide, which has at
least 80%, preferably 90%, even more preferably 95% amino acid
sequence identity with the "fragment of a recombinant protein" or
"fragment of a coat protein", respectively, as defined above and
which is preferably capable of assembling into a virus-like
particle.
[0063] The term "mutant recombinant protein" or the term "mutant of
a recombinant protein" as interchangeably used in this invention,
or the term "mutant coat protein" or the term "mutant of a coat
protein", as interchangeably used in this invention, refers to a
polypeptide having an amino acid sequence derived from the wild
type recombinant protein, or coat protein, respectively, wherein
the amino acid sequence is at least 80%, preferably at least 85%,
90%, 95%, 97%, or 99% identical to the wild type sequence and
preferably retains the ability to assemble into a VLP.
[0064] The invention is related to an efficient fermentation
process for the production of a VLP of a bacteriophage. The process
is improved with respect to yield of the VLP and can be scaled up
to a commercial production scale. The process encompasses the
expression of recombinant capsid protein of bacteriophages in a
bacterial host under conditions which allow the capsid protein to
self-assemble into VLPs spontaneously.
[0065] Specific examples of VLPs which can be produced by the
process of the invention are VLPs of bacteriophages, preferably RNA
bacteriophages. In one preferred embodiment of the invention, the
virus-like particle of the invention comprises, consists
essentially of, or alternatively consists of, recombinant coat
proteins, mutants or fragments thereof, of a RNA-phage. Preferably,
the RNA-phage is selected from the group consisting of a)
bacteriophage Q.beta.; b) bacteriophage R17; c) bacteriophage fr;
d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g)
bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k)
bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage
AP205.
[0066] In one preferred embodiment of the invention, VLPs are
produced comprising coat protein, mutants or fragments thereof, of
RNA bacteriophages, wherein the coat protein has an amino acid
sequence selected from the group consisting of: (a) SEQ ID NO:5
referring to Q.beta. CP; (b) a mixture of SEQ ID NO:5 and SEQ ID
NO:15 (Q.beta. A1 protein); (c) SEQ ID NO:16 (R17 capsid protein);
(d) SEQ ID NO:17 (fr capsid protein); (e) SEQ ID NO:18 (GA capsid
protein); (f) SEQ
[0067] ID NO:19 (SP capsid protein); (g) a mixture of SEQ ID NO:19
and SEQ ID NO:20; (h) SEQ ID NO:21 (MS2 capsid protein); (i) SEQ ID
NO:22 (M11 capsid protein); (j) SEQ ID NO:23 (MX1 capsid protein);
(k) SEQ ID NO:24 (NL95 capsid protein); (1) SEQ ID NO:25 (f2 capsid
protein); (m) SEQ ID NO:26 (PP7 capsid protein); and (n) SEQ ID
NO:12 (AP205 capsid protein).
[0068] Upon expression in E. coli, the N-terminal methionine of
Q.beta. coat protein is usually removed (Stoll, E. et al., J. Biol.
Chem. 252:990-993 (1977)). VLP composed of Q.beta. coat proteins
where the N-terminal methionine has not been removed, or VLPs
comprising a mixture of Q.beta. coat proteins where the N-terminal
methionine is either cleaved or present are also within the scope
of the present invention.
[0069] In one preferred embodiment of the invention, the VLP is a
mosaic VLP comprising or alternatively consisting of more than one
amino acid sequence, preferably two amino acid sequences, of coat
proteins, mutants or fragments thereof, of a RNA bacteriophage.
[0070] In one very preferred embodiment, the VLP comprises or
alternatively consists of two different coat proteins of a RNA
bacteriophage, said two coat proteins have an amino acid sequence
of SEQ ID NO: 5 and SEQ ID NO:15, or of SEQ ID NO:19 and SEQ ID
NO:20.
[0071] In preferred embodiments of the present invention, the
produced VLP comprises, or alternatively consists essentially of,
or alternatively consists of recombinant coat proteins, mutants or
fragments thereof, of the RNA-bacteriophage Q.beta., fr, AP205 or
GA.
[0072] In one preferred embodiment, the VLP is a VLP of RNA-phage
Q.beta.. The capsid or virus-like particle of Q.beta. shows an
icosahedral phage-like capsid structure with a diameter of 25 nm
and T=3 quasi symmetry. The capsid contains 180 copies of the coat
protein, which are linked in covalent pentamers and hexamers by
disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554
(1996)).
[0073] Preferred virus-like particles of RNA bacteriophages, in
particular of Q.beta. and fr in accordance of this invention are
disclosed in WO 02/056905, the disclosure of which is herewith
incorporated by reference in its entirety. Particular Example 18 of
WO 02/056905 gave detailed description of preparation of VLP
particles from Q.beta..
[0074] In another preferred embodiment, the VLP is a VLP of RNA
bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs,
including AP205 coat protein with the substitution of proline at
amino acid 5 to threonine, may also be used in the practice of the
invention and leads to other preferred embodiments of the
invention. WO 2004/007538 describes, in particular in Example 1 and
Example 2, how to obtain VLP comprising AP205 coat proteins, and
hereby in particular the expression and the purification thereto.
WO 2004/007538 is incorporated herein by way of reference.
[0075] In one preferred embodiment, the VLP comprises or consists
of a mutant coat protein of a virus, preferably a RNA
bacteriophage, wherein the mutant coat protein has been modified by
removal of at least one lysine residue by way of substitution
and/or by way of deletion. In another preferred embodiment, the VLP
of the invention comprises or consists of a mutant coat protein of
a virus, preferably a RNA bacteriophage, wherein the mutant coat
protein has been modified by addition of at least one lysine
residue by way of substitution and/or by way of insertion. The
deletion, substitution or addition of at least one lysine residue
allows varying the degree of coupling with an antigen.
[0076] VLPs or capsids of Q.beta. l coat protein display a defined
number of lysine residues on their surface, with a defined topology
with three lysine residues pointing towards the interior of the
capsid and interacting with the RNA, and four other lysine residues
exposed to the exterior of the capsid.
[0077] Q.beta. mutants, of which exposed lysine residues are
replaced by arginines are also encompassed by the present
invention. Preferably these mutant coat proteins comprise or
alternatively consist of an amino acid sequence selected from the
group of a) Q.beta.-240 (SEQ ID NO:7, Lys13.fwdarw.Arg); b)
Q.beta.-243 (SEQ ID NO:8, Asn10.fwdarw.Lys); c) Q.beta.-250 (SEQ ID
NO:9, Lys2.fwdarw.Arg); d) Q.beta.-251 (SEQ ID NO:10,
Lys16.fwdarw.Arg); and e) Q.beta.-259 (SEQ ID NO:11,
Lys2.fwdarw.Arg, Lys16.fwdarw.Arg). The construction, expression
and purification of the above indicated Q.beta. mutant coat
proteins, mutant Q.beta. coat protein VLPs and capsids,
respectively, are described in WO02/056905. In particular is hereby
referred to Example 18 of above mentioned application.
[0078] In a further preferred embodiment the recombinant capsid
protein is a capsid protein of bacteriophage AP205 having the amino
acid sequence depicted in SEQ ID NO:12 or a mutation thereof, which
is capable of forming a VLP, for example the proteins AP205P5T (SEQ
ID NO:13) or AP205 N14D (SEQ ID NO:14.).
[0079] In a very preferred embodiment said recombinant capsid
protein is composed of the 133 amino acid coat protein C of E. coli
RNA bacteriophage Q.beta. comprising or preferably consisting of
the amino acid sequence depicted in SEQ ID NO:5, wherein preferably
said recombinant capsid protein is capable of forming a VLP by
self-assembly.
[0080] In one embodiment, the expression construct comprises a
first stop codon and a second stop codon, wherein said first stop
codon is located directly 3' of said first nucleotide sequence and
wherein said second stop codon is located directly 3' of said first
stop codon, and wherein at least one of said first or second stop
codon is TAA. For example, plasmid pTac-nSDAP205 (SEQ ID NO:30)
comprises the naturally occurring TAA stop codon as a first stop
codon and an additional TGA stop codon directly 3' of the first
stop codon.
[0081] In a preferred embodiment the expression construct comprises
a first nucleotide sequence and a second nucleotide sequence,
wherein said first nucleotide sequence is encoding a recombinant
capsid protein, preferably Q.beta. CP, or a mutant or fragment
thereof, most preferably SEQ ID NO:5, and wherein said second
nucleotide sequence is encoding any other protein, preferably the
Q.beta. A1 protein or a mutant or fragment thereof, most preferably
SEQ ID NO:15, and wherein said first and said second nucleotide
sequence are separated by exactly one sequence stretch comprising
at least one TAA stop codon. In one embodiment said TAA stop codon
is generated by replacing the naturally occurring stop codon,
preferably TGA by the sequence TAA. Alternatively and more
preferably said TAA stop codon is generated by replacing the
naturally occurring stop codon, preferably TGA by the sequence
TAATGA (SEQ ID NO:32).
[0082] For example, the region of Q 2 gene C corresponds to the
NCBI GenBank Acc. No. M99039 (nucleotides 46-1062). Gene C contains
a first nucleotide sequence encoding the 133-amino acid Q.beta.
coat protein (SEQ ID NO:5) and a second nucleotide sequence
encoding the 329-amino acid read through protein A1 (SEQ ID NO:15).
Nucleotides 1-399 of SEQ ID NO:6 (nucleotides 46-444 of NCBI
GenBank Acc. No. M99039) correspond to said first nucleotide
sequence encoding the 133-amino acid Q.beta. CP, Nucleotides 400 to
402 of SEQ ID NO:6 correspond to the strong TAA stop codon and
nucleotides 403 to 405 of SEQ ID NO:6 to the leaky TGA stop codon,
which is followed by said second nucleotide sequence (Q.beta. A1).
Surprisingly, it was found that the presence of the nucleotide
sequence relating to A1 in the expression construct results in
higher RNA stability and, thus, in improved yield of Q.beta. CP and
VLP as compared to a construct wherein the A1 sequence is
deleted.
[0083] The expression of a recombinant protein can significantly
reduce the growth rate of the bacterial host due to toxic effects
of the accumulating protein and due to the metabolic burden caused
by the protein synthesis. In particular cell lysis and low plasmid
retention may occur. Inducible promoters provide for the
possibility to separate the growth phase from the production phase
of a fermentation process. Inducible promoters are repressed by a
repressor molecule during the growth phase of the bacterial host
and are induced by exposing the bacterial host to inductive
conditions during the production phase. Inducible promoters
therefore allow the bacterial host to grow fast, preferably
exponentially during the growth phase and to reach high cell
densities. Thus, inducible promoters provide for high yield of the
expression product at the end of the production phase. Therefore,
the usage of inducible promoters for the expression of recombinant
protein is preferred.
[0084] A well known example for an inducible promoter is the lac
promoter which forms part of the lac operon and which can be
induced by addition of lactose or the strong synthetic inducer
isopropylthio-.beta.-D-galactosid (IPTG) to the growth medium of
the bacterial host. Donavan et al. 2000 (Can. J. Microbiol
46:532-541) report on an improved process for the expression of a
monoclonal antibody fragment under the control of the lac promoter.
Further examples of inducible promoters are provided in table 1 of
Makrides 1996 (Microbiological Reviews, p. 512-538).
[0085] A typical drawback of expression systems based on inducible
promoters is the "leakiness" of the promoter, meaning that the
promoter is only insufficiently repressed and causes a certain
expression rate of the recombinant protein during the growth phase.
This typically leads to a reduced cell density or to plasmid
instability and, as a consequence, to reduced yield of the
recombinant protein Makrides 1996 (Microbiological Reviews, p.
512-538). An example of a promoter which is prone to insufficient
repression is the VHb promoter which is repressed under high oxygen
conditions and induced upon oxygen depletion.
[0086] For the purpose of the invention promoters are preferred
which are stringently repressed. In one embodiment the promoter is
repressed by the repressor lacI. Examples of such promoters are
disclosed in Makrides 1996 (Microbiol. Rev. 60:512-538), Goldstein
& Doi 1995 (Biotechnology Annual Review 1:105-128), Hannig
& Makrides 1998 (TIBTECH 16:54-60) and Stevens 2000 (Structures
8, R177-R185). In a preferred embodiment the promoter is inducible
by lactose, more preferably it is selected from the group
consisting of lac, lacUV5, tac, trc, P.sub.syn lpp.sup.a, lpp-lac,
T7-lac, T3-lac, and T5-lac. Especially preferred for the purpose of
the invention is the tac promoter (SEQ ID NO:2) or a mutation or
variant thereof. Within the scope of the invention are mutants or
truncated or deleted variants of the tac promoter having a sequence
homology with SEQ ID NO:2 which is at least 50%, 60%, 70%, 80, 90,
or 95%, preferably 98 to 100%, most preferably 99%. Wherein the
promoter strength of such mutated truncated or deleted variant is
comparable to that of the promoter of SEQ ID NO:2. The skilled
person will be able to determine the promoter strength of a given
sequence by comparative expression studies using standard methods.
In a specific embodiment of the invention the promoter driving the
expression of the recombinant capsid protein comprises or
alternatively consists of SEQ ID NO:2. The tac promoter is a fusion
product of the -10 region of the lacUV5 promoter and the -35 region
of the trp promoter and combines the high transcription efficiency
of trp with the regulatory elements of the lac promoter (de Boer et
al. 1983, PNAS 80:21-25; Amann et al. 1983 Gene 25:167-178). It
provides for sufficiently high expression rates and high protein
yield while avoiding the formation of insoluble or incorrectly
folded recombinant protein which may occur with stronger promoters,
such as the T7 promoter. The trc and the tic promoter are mutated
versions of the tac promoter (Brosius et al. 1985, The Journal of
Biological Chemistry 260(6):3539-3541). In a further preferred
embodiment the promoter is selected from the group consisting of
tic, trc and tac.
[0087] For the construction of an expression construct for the
purpose of the invention the promoter is operably linked to said
first nucleotide sequence encoding the recombinant capsid protein
via a ribosome binding site (Shine-Dalgarno sequence, SD),
typically comprising an
[0088] ATG start codon at its 3' end. Suitable Shine-Dalgarno
sequences for the purpose of the invention are well known in the
art (Dalboge et al. 1988, DNA 7(6):399-405; Ringquist et al. 1992,
Mol. Micr. 6:1219-1229). In one embodiment of the invention the
expression construct comprises the SD sequence of Dalboge et al.
1988 (DNA 7(6):399-405) which is depicted in SEQ ID NO:4. In
another, preferred, embodiment the expression construct comprises a
Shine-Dalgamo sequence of Ringquist et al. 1992 (Mol. Micr.
6:1219-1229, SEQ ID NO:3, nSD). Surprisingly, it was found that SEQ
ID NO:3 is particularly suited for the purpose of the invention
because it results in improved expression levels and improved yield
of recombinant capsid protein. SEQ ID NO:3 is especially suited to
enhance the expression of AP205 capsid protein. In a preferred
embodiment of the invention the expression construct comprises a
Shine-Dalgamo sequence selected form the group consisting of SEQ ID
NO:3 and SEQ ID NO:4, preferably said Shine-Dalgarno sequence is
SEQ ID NO:3.
[0089] Transcriptional terminators are functional elements of
expression constructs. The skilled person will be able to choose a
suitable terminator sequence form a wide range of sources. In a
preferred embodiment of the invention said expression construct
comprises a terminator sequence, wherein preferably said terminator
sequence is operably linked to said first nucleotide sequence,
wherein further preferably said terminator sequence is the rRNB
terminator sequence, most preferably SEQ ID NO:28.
[0090] For the purpose of plasmid selection the skilled person will
typically use an antibiotic resistance marker gene. Examples of
antibiotic resistance genes which are widely used in the art and
which are suitable for the purpose of the invention are resistance
genes against the antibiotics ampicillin, tetracyclin and
kanamycin. The use of kanamycin as a selective agent in the frame
of a process for the production of a VLP is generally preferred
because of the lower allergenic potential of kanamycin as compared
to alternative antibiotics and because of the lower safety concerns
resulting thereof for the use of the VLP as a vaccine. Furthermore,
kanamycin provides better plasmid retention as compared to
alternative antibiotics such as ampicillin. The kanamycin
3'-phosphotransferase gene (SEQ ID NO:29) which is derived from the
transposon Tn903 is therefore a particularly useful selectable
marker gene.
[0091] The addition of antibiotics to the medium is generally
undesirable in a commercial production process for cost and safety
reasons. In the context of the invention antibiotics, preferably
kanamycin, are typically and preferably used for the selection of
the expression strain. Media used in the production process are
essentially free of antibiotics, in particular kanamycin. However,
addition of an antibiotic to precultures used to produce the
inoculum for the production culture can improve plasmid retention
throughout the process (Example 10).
[0092] The skilled person will create expression plasmids
comprising expression constructs which are useful for the
production of VLPs of bacteriophages by combining the genetic
elements described above applying standard methods of molecular
biology. Particularly useful expression plasmids for the purpose of
the invention are pTac-nSDQb-mut (SEQ ID NO:1) for the production
of Q.beta. VLP and pTac-nSDAP205 (SEQ ID NO:30) for the production
of AP205 VLP. The construction of these specific expression
plasmids is described in detail in the Examples section.
[0093] The expression plasmids are transformed to a bacterial
expression host by any method known in the art, preferably by
electroporation. Individual clones of the host comprising the
expression plasmid are selected for maximal expression of the
recombinant capsid protein by SDS-PAGE after cell lysis. Selected
clones of the expression host comprising the expression plasmid can
be stored as frozen glycerol cultures.
[0094] Said bacterial host can be chosen from any bacterial strain
capable of replicating and maintaining said expression plasmid
during cell division. Preferred bacterial hosts are Escherichia
coli strains having the specific features described in the
following sections.
[0095] The repression of the promoter is improved by overexpression
of the repressor by the bacterial host. In one embodiment said
cultivating of said bacterial host is performed in batch culture
and under conditions under which said promoter is repressed by lad.
In a preferred embodiment the gene causing overexpression of said
lad in said bacterial host is located on a plasmid, preferably on
said expression plasmid. Alternatively, said gene is located on a
separate plasmid contained in said bacterial host, wherein said
separate plasmid preferably is a high copy number plasmid.
Alternatively, and most preferably said gene is located on the
chromosome of said bacterial host.
[0096] One example of a gene causing overexpression of lacI is
lacI.sup.q (Menzella et al. 2003, Biotechnology and Bioengineering
82(7)809-817) which is a single CG to TA change at -35 of the
promoter region of lacI which causes a 10 fold increase in LacI
expression. A further example is lacIQ1 (Glascock & Weickert
1998, Gene 223(1-2):221-231). Improved repression of the promoter
during the growth phase results in improved plasmid retention and
higher cell density and, ultimately, in improved protein yield. For
example, bacterial strains comprising the lacIq gene overexpress
the lad repressor molecule and therefore prevent formation of the
recombinant protein during the growth phase more efficiently than
strains comprising the wildtype gene. In a preferred embodiment the
gene causing overexpression of said lad is lacIQ1 or lacI.sup.q,
preferably lacI.sup.q. In a specifically preferred embodiment said
bacterial host comprises the lacI.sup.q gene on its chromosome.
[0097] In one embodiment said inducing of said promoter is
performed with an inducer, wherein said inducer is preferably
selected from IPTG and lactose, most preferably said inducer is
lactose. Upon exposure of the bacterial host to an inducer, the
repressor is inactivated and the promoter becomes active. Addition
of the strong inducer IPTG to the culture medium results in an
immediate increase of the expression rate of the recombinant
protein to a high level because IPTG directly enters the cells by
diffusion and binds and inactivates the active repressor lad.
Inactivated lacI repressor molecules dissociates from the operator
and allow high level transcription from the promoter. IPTG is not
metabolized by the cell and the transcription continues with high
rates until other metabolic parameters become limiting.
[0098] As mentioned before, high expression rates may lead to the
formation of insoluble recombinant protein which is not capable of
forming a VLP by self-assembly. Induction of protein expression
with high concentrations of IPTG is particularly prone to the
formation of insoluble protein. Therefore, induction of the
promoter is preferably achieved by the addition of IPTG in
concentrations which are below the concentration which causes the
expression to occur at its maximum rate (Kopetzki et al. 1989, Mol
Gen Genet 216:149-155).
[0099] In a preferred embodiment said inducing of said promoter is
performed with IPTG, wherein the concentration of said IPTG in said
medium is about 0.001 to 5 mM, preferably 0.001 to 1 mM, more
preferably 0.005 to 1 mM, still more preferably 0.005 to 0.5 mM. In
a specifically preferred embodiment the concentration of said IPTG
is about 0.01 mM, most preferably 0.01 mM.
[0100] Alternatively, induction of the promoter is achieved by the
addition of lactose. Induction of recombinant protein expression
with lactose requires that the bacterial host is capable of taking
up lactose from the medium, e.g. by Lac permease and that it
comprises .beta.-galactosidase activity. The Lac permease dependent
uptake of lactose into the cells follows a slower kinetic than the
uptake of IPTG by diffusion. Furthermore, lactose does not directly
interact with the lac operon but is converted by
.beta.-galactosidase to allolactose
(1-6-O-.beta.-galactopyranosyl-D-glucose) which is the actual
inducer of the promoter. Induction of recombinant protein
expression by the addition of lactose is advantageous because it
avoids the immediate increase of the expression rate to a maximum
level upon addition of the inducer and, thus, it reduces the risk
of the formation of insoluble protein.
[0101] Allolactose is metabolised by the bacterial host during the
production phase and contributes carbon and metabolic energy to the
bacterial metabolism. This may further contribute to improved
protein yield as compared to induction with IPTG. Furthermore,
induction by lactose allows to a certain extend the control of the
expression rate of the recombinant protein during the production
phase via the lactose concentration in the medium. Induction by
lactose is further preferred in a pharmaceutical production process
because IPTG is expensive and is believed to be toxic. Its removal
needs to be demonstrated at the end of a the production
process.
[0102] In a preferred embodiment said inducing of said promoter is
performed by the addition of lactose to said batch culture, wherein
preferably said bacterial host is capable of taking up lactose from
the medium and wherein further preferably said bacterial host
comprises .beta.-galactosidase activity. Such bacteria strains can,
for example, be obtained from strain collections such as ATTC
(http://www.atcc.org). In a preferred embodiment, said bacterial
host is an E. coli strain, preferably an E. coli strain selected
from the group consisting of RB791, DH2O, Y1088, W3110 and MG1655.
Most preferably, said bacterial host is E. coli RB791. In a still
more preferred embodiment said promoter is the tac promoter or
mutant or variant thereof and said bacterial host is an E. coli
strain which further comprises a gene causing overexpression of a
repressor of the tac promoter, wherein said gene preferably is
lacI.sup.q. The pH of the culture medium of the bacterial host can
be controlled during the fermentation process and regulated by the
addition of acidic or alkaline solutions using methods which are
well known in the art. In one embodiment, said cultivating of said
bacterial host and said feeding of said batch culture is performed
under conditions, wherein the pH of the medium is controlled. In a
preferred embodiment said pH is between 5.5 and 8.0, more
preferably between 6.5 and 7.5, even more preferably between 6.7
and 7.0 and most preferably said pH of said medium is 6.8. Said pH
of said medium may be kept constant during the process or it may
follow a certain profile during the different phases of the process
within the pH ranges specified above. In a preferred embodiment
said pH is kept constant at a value of 6.7 to 7.0, preferably said
pH it is kept constant at 6.8.
[0103] The process of the invention comprises a growth phase,
wherein said growth phase comprises a batch and a feed phase,
wherein said growth phase and simultaneously said batch phase are
initiated by said cultivating said bacterial host and wherein said
feed phase is initiated by said feeding of said batch culture with
said major carbon source.
[0104] The oxidative capacity of bacteria cells is limited and high
concentrations of the substrate may cause the formation of reduced
products like acetate, which may lead to undesired acidification of
the medium and to reduced growth of the bacteria. Therefore, the
bacterial host is grown in a fed-batch culture on a minimum medium
with a limited quantity of substrate. In one embodiment said
cultivating of said bacterial host is performed in a medium
comprising said major carbon source, wherein said medium preferably
is a minimal medium, preferably a chemically defined minimal
medium. Most preferably said medium is R27 medium as described in
Example 5.
[0105] At the end of the batch phase, when the substrate contained
in the medium is almost exhausted, medium containing the major
carbon source (feed medium) is fed to said batch culture at the
same rate as the desired growth rate of the bacterial host, i.e.
the growth rate of the bacterial host is limited by the feed rate
of the substrate. It is understood by the skilled person that the
decisive parameter is the actual mass flow of the substrate,
preferably the major carbon source, and other nutrients required to
maintain growth. Since in practice a constant composition of the
feed medium can be assumed, the flow rate refers to the volume flow
of the medium. The same consideration applies to the co-feed medium
(see below).
[0106] Therefore, in one embodiment said feeding of said batch
culture with said major carbon source is performed with a flow
rate, wherein said flow rate is limiting the growth rate of said
bacterial host.
[0107] During the feed phase the growth rate can be freely selected
in a wide range nearly up to the maximum growth rate (.mu..sub.max)
if no inhibition occurs. The actual value of .mu..sub.max is highly
dependent on the bacterial strain, the expression construct and the
growth conditions. The skilled person will understand that the
determination of .mu..sub.max is performed under conditions under
which the promoter is repressed.
[0108] For a given experimental set up, .mu. can be determined from
the growth curve of the culture by plotting biomass concentration
(x) as determined by OD.sub.600 or cell wet weight (CWW) against
the cultivation time and determining the exponential growth
coefficient .mu. based on the equation x=x.sub.0 e.sup..mu.t. The
actual value of .mu..sub.max is determined as the growth rate .mu.
of an exponentially growing batch culture in the beginning of the
batch phase when no substrate limitation occurs, i.e. without
supply of additional medium by feeding. The growth rate .mu. can be
determined by computing the ratio of the difference between natural
logarithm of the total biomass X.sub.2 measured at time t.sub.2 and
natural logarithm of the total biomass X.sub.1 measured at time
t.sub.1 to the time difference (t.sub.2-t.sub.1):
.mu.=(lnX.sub.2-lnX.sub.1)/(t.sub.2-t.sub.1).
[0109] Fed-batch culture allows the maintenance of a constant
growth rate (.mu.). In a preferred embodiment the substrate,
preferably the major carbon source, is fed during the feed phase
according to the exponential increase of the biomass (x). If during
the feed phase the substrate is supplied at the same rate it is
consumed, the culture is in a quasi steady state, analogous to the
cultivation in a continuous culture. Because biomass formation and
substrate consumption are correlated over the substrate-referred
yield coefficient Y.sub.x/s (biomass [g]/substrate [g]), the
substrate quantity (s) per time unit (t) to be supplied is
calculated according to the formula ds/dt=.mu./Y.sub.x/x x.sub.0
tote.sup..mu.t, wherein x.sub.0 tot is the total biomass at feed
start.
[0110] Therefore, in a preferred embodiment said feeding of said
batch culture with said major carbon source is performed with a
flow rate, wherein said flow rate increases with an exponential
coefficient .mu., and wherein preferably said exponential
coefficient .mu. is below .mu.max. Thus, the growth rate of said
bacterial host during the feed phase is set to a value which is
below .mu..sub.max. In a preferred embodiment said exponential
coefficient .mu. is about 30% to 70%, most preferably about 50% of
.mu..sub.max. In a specific embodiment of the invention .mu. is set
to an absolute value of 0.15 to 0.45 h.sup.-1, more preferably 0.25
to 0.35 h.sup.-1, most preferably .mu. is 0.3 h.sup.-1, provided
that the set up of the process is such, that these values are below
.mu..sub.max.
[0111] Bacteria are able to utilise a wide range of different
substrates. For the purpose of the invention, preferred major
carbon sources are glucose and glycerol, preferably glycerol.
Although the maximum specific growth rate (.mu..sub.max) of the
expression host which can be achieved may be higher with glucose
than with glycerol, glycerol causes less acetate formation and
provides higher biomass yield per substrate (Y.sub.x/s) and,
ultimately, higher yield of the recombinant protein. Furthermore,
the handling of the liquid substrate glycerol is easier than that
of solid carbon sources like glucose which need to be dissolved in
a separate process step.
[0112] As mentioned before, plasmid retention, i.e. the maintenance
of the expression plasmid in the bacterial host during the
fermentation process, is essential for optimal yield of the
recombinant protein. Plasmid retention can be assessed by spreading
bacteria cells on a solid medium to form single colonies and
testing individual colonies for their antibiotic resistance. For
example, a plasmid retention of 100% means that 100 out of 100
tested colonies comprise the specific antibiotic resistance which
conferred by the expression plasmid. For the purpose of the
invention plasmid retention at the end of the fermentation process
is more than 80%, preferably more than 90%, more preferably more
than 95%, even more preferably more than 97% and most preferably
100%.
[0113] The optimal growth temperature of a bacterial strain is the
temperature at which it reaches its highest maximal growth rate
(.mu..sub.max). Under otherwise not limiting conditions for most E.
coli strains this temperature is about 37.degree. C. However,
growth of the bacterial strain comprising the expression construct
at the optimal growth temperature and in the absence of a selective
antibiotic may favour the loss of the expression plasmid, whereas
plasmid retention is generally improved when the expression strain
is grown at lower temperature. Although the maximum growth rate of
the expression strain is lower when the strain is grown at
temperatures below its optimal growth temperature as compared to
growth at the optimal growth temperature, the yield of recombinant
protein may be equal or even better at the lower temperature due to
improved plasmid retention.
[0114] In one embodiment of the invention, said cultivating of said
bacterial host and/or said feeding of said batch culture with said
major carbon source and/or said inducing said promoter with an
inducer is therefore performed at a temperature below the optimal
growth temperature of said bacterial host. In a preferred
embodiment said temperature is between 20 and 37.degree. C.,
preferably between 23 and 35.degree. C., more preferably between 25
and 33.degree. C., even more preferably between 27 and 32.degree.
C., still more preferably between 28 and 31.degree. C. Still more
preferably said temperature is about 30.degree. C., most preferably
said temperature is 30.degree. C.
[0115] The process of the invention comprises a production phase,
wherein said production phase is initiated by said inducing said
promoter with an inducer. The time point for the initiation of said
production phase can be determined based on cultivation time and/or
growth parameters.
[0116] The growth of the bacterial host during the fermentation
process can be assessed by determining the optical density at 600
nm (OD.sub.600), the cell wet weight (CWW [g/l]) and the cell dry
weight (CDW [g/l]). These parameters can be used to define the
optimal time point for the start of the production phase by
addition of the inducer, preferably lactose, to the medium. It is
apparent for the skilled person, that on one hand higher CWW at the
beginning of the production phase can be achieved by an extended
feed phase and may lead to improved yield of the recombinant
protein but that on the other hand over-aged cultures may show
insufficient protein expression. The optimal time point for the
beginning of the production phase, which is initiated by said
inducing of said promoter with an inducer, therefore needs to be
determined for the specific production conditions. For example, for
expression of Q.beta. CP in E. coli RB791 in a total volume of 2 l,
induction is started after ca. 14 h, when OD.sub.600 has reached
about 40 to 60. Surprisingly, similar parameters were found for the
same process in a 50 l scale, where induction start is also after
ca. 14 h when OD.sub.600 has reached about 50.
[0117] Therefore, in one embodiment of the invention, said inducing
of said promoter with said inducer is performed 10 h to 16 h after
the beginning of said growth phase, preferably after 12 h to 15 h,
more preferably after 13 h to 15 h, most preferably after about 14
h, wherein preferably said inducing of said promoter with said
inducer is performed when the OD.sub.600 has reached about 40 to
60, preferably about 50.
[0118] In a further embodiment, said inducing of said promoter with
said inducer is performed after an extended feed phase, wherein
preferably said inducing of said promoter with said inducer is
performed 14 h to 20 h after the beginning of said cultivating of
said bacterial host in a medium, preferably after 15 h to 18 h,
more preferably after 16 h to 17 h, most preferably after about
16.5 h, wherein preferably said inducing of said promoter with said
inducer is performed when the OD.sub.600 has reached about 80 to
90, preferably about 85.
[0119] In one embodiment of the invention said inducing of said
promoter with said inducer is performed when the OD.sub.600 reached
a value of 25 to 60, preferably 25 to 55, more preferably 30 to 50,
most preferably 30 to 40. In a specifically preferred embodiment
said inducing of said promoter with said inducer is performed when
OD.sub.600 is 35.
[0120] In another embodiment of the invention said inducing of said
promoter with said inducer is performed after an extended feed
phase, when the OD.sub.600 reached a value of 60 to 120, preferably
70 to 110, more preferably 80 to 100, most preferably 80 to 90. In
a specifically preferred embodiment the induction is started after
and an extended feed phase when OD.sub.600 is about 85, preferably
85.
[0121] Induction with IPTG: In one embodiment of the invention said
inducing of said promoter with an inducer is achieved by the
addition of IPTG, wherein preferably said feeding of the culture
with the major carbon source is continued. Since IPTG is not
metabolized by the bacterial host, induction can be achieve by a
single addition of IPTG to the desired concentration.
Alternatively, induction can be achieved by a continuous flow of
IPTG to the culture. In a preferred embodiment induction is
performed by addition of IPTG in a single addition or a continuous
flow, wherein said feeding of the batch culture with the major
carbon source is continued with a constant or an increasing flow
rate of said major carbon source exponentially increasing flow rate
of the major carbon source.
[0122] Induction with lactose: As described above, the induction of
protein expression can alternatively be achieved by the addition of
lactose to the culture medium. In one embodiment of the invention,
at the beginning of the production phase the exponential feed of
the substrate is interrupted and the culture is supplied with a
constant flow of induction medium containing 100 to 300 g/l,
preferably 100 g/l lactose as the sole carbon source (lactose feed
medium). Preferably, the constant flow rate of lactose equals
approximately the flow rate of the substrate at the end of the feed
phase.
[0123] In a preferred embodiment of the invention said inducing of
said promoter with an inducer is achieved by the addition of
lactose, wherein preferably said lactose is fed to said batch
culture in a continuous flow during and wherein preferably said
feeding of said batch culture with said major carbon source is not
continued.
[0124] Upon addition of lactose to the culture, the
f3-galactosidase activity increases, lactose is converted to
allolactose which induces the tac promoter and the expression of
the recombinant capsid is initiated. In parallel, allolactose is
further metabolised and contributes to the energy supply for the
bacterial host. The equilibrium of the feeding rate of the
induction medium and the lactose consumption by the cells thus
determines the expression rate. The enzymatic reactions involved in
this cascade allow to control the process in such a way that the
formation of inclusion bodies is minimised. The progress of
induction process can be monitored by determining the
.beta.-galactosidase activity in the culture, e.g. by a .beta.-Gal
Assay Kit (Invitrogen, K1455-01).
[0125] In a more preferred embodiment of the invention said
inducing of said promoter with an inducer is achieved by the
addition of lactose, wherein preferably said lactose is fed to said
batch culture in a continuous flow during and wherein preferably
said feeding of said batch culture with said major carbon source is
continued.
[0126] Discontinuous addition of inducer: Said inducer can be added
to the culture discontinuously by a single addition at the
beginning of the production phase or by a few subsequent additions
during the production phase. Discontinuous addition of the inducer,
especially by a single addition is particularly suited when the
inducer is IPTG since IPTG is not metabolized by the bacterial
host. Therefore, typically and preferably no replacement of
metabolised IPTG is necessary during the production phase. In one
embodiment said inducing of said promoter with an inducer is
performed by the addition of said inducer, preferably IPTG or
lactose, most preferably IPTG, to said medium, wherein said inducer
is added to about its final concentration at once by a single
addition at the beginning of the production phase, wherein
preferably said feeding of said batch culture with said major
carbon source is continued. In a preferred embodiment said inducing
of said promoter with an inducer is performed by the addition of
IPTG to said medium, wherein said IPTG is added to about its final
concentration at once by a single addition, wherein preferably said
feeding of said batch culture with said major carbon source is
continued. Alternatively, said inducing of said promoter with an
inducer is performed by the addition of said inducer, preferably
IPTG or lactose, most preferably lactose, to said medium, wherein
said addition is performed in several steps, preferably in 1 to 5,
more preferably in 2 to 4, most preferably in 3 steps during the
production phase, wherein preferably said feeding of said batch
culture with said major carbon source is continued.
[0127] Continuous addition (feeding) of inducer: Preferably, said
inducer is added to the medium in a continuous flow, preferably
throughout the production phase. The continuous addition of the
inducer is particularly suited for lactose, since lactose is
metabolised by the bacterial host and therefore a continuous
addition of lactose during the production phase allows to maintain
a lactose concentration in the medium which allows for efficient
induction of the promoter. In a preferred embodiment, said inducing
of said promoter with an inducer is performed by feeding said batch
culture with said inducer, wherein preferably said inducer is IPTG
or lactose, most preferably lactose, and wherein said feeding is
performed in a continuous flow, wherein further preferably said
feeding is performed throughout the production phase.
[0128] Co-feeding of inducer and major carbon source: The
expression of the recombinant protein is an energy demanding
process. To prevent yield loss which might be caused by the
excessive consumption of the inducer by the bacterial host and low
expression rates resulting thereof, the culture can be additionally
supplemented with substrate, preferably the major carbon source,
during the production phase, wherein the flow rate of inducer
and/or the major carbon source is constant or increasing,
preferably constant. When during the production phase the culture
is supplemented with substrate at an increasing flow rate, the flow
rate is preferably increasing with an exponential rate.
[0129] Co-feeding with constant flow rate: In a preferred
embodiment said inducing of said promoter with an inducer is
performed by co-feeding said batch culture with said inducer and
said major carbon source, wherein said inducer is preferably IPTG
or lactose, most preferably lactose, and wherein said major carbon
source is glucose or glycerol, preferably glycerol, wherein said
inducer, preferably lactose and said major carbon source,
preferably glycerol are co-fed to said batch culture at a flow
rate, wherein said flow rate is preferably about constant. In a
further preferred embodiment said flow rate is chosen to allow
feeding of said major carbon source to said batch culture at about
the same rate as at the end of the growth phase. In a still further
preferred embodiment said inducer, preferably lactose, and said
major carbon source, preferably glycerol, are contained in the same
medium (co-feed medium). In a further preferred embodiment said
co-feed medium is fed to said batch culture with a flow rate,
wherein said flow rate is preferably about constant, and wherein
further preferably said flow rate is chosen to allow feeding of
said major carbon source to said batch culture at about the same
rate as at the end of the growth phase. In a very preferred
embodiment said inducer is lactose and said major carbon source is
glycerol, wherein said lactose and said glycerol are co-fed to said
batch culture in a ratio of about 2:1 to 1:4 (w/w).
[0130] In a further preferred embodiment of the invention lactose
and said major carbon source, preferably glycerol, are co-fed to
said batch culture in a ration of 0:1 to 1:0 (w/w), preferably
about 2:1 to about 1:4 (w/w), more preferably about 1:1 to 1:3
(w/w), most preferably the ratio is about 1:3 (w/w). In a preferred
embodiment the ratio of lactose and the major carbon source,
preferably glycerol, is 1:1 (w/w). In another preferred embodiment
the ratio of lactose and the major carbon source, preferably
glycerol, is 1:3 (w/w). In a more preferred embodiment said co-feed
medium comprises ca. 200 g/l lactose and ca. 200 g/l glycerol. In a
still more preferred embodiment the co-feed medium comprises ca.
100 g/l lactose and ca. 300 g/l glycerol.
[0131] Co-feeding with increasing flow rate: Alternatively, said
inducing of said promoter with an inducer is performed by
co-feeding said batch culture with said inducer and said major
carbon source, wherein said inducer is preferably IPTG or lactose,
most preferably lactose, and wherein said major carbon source is
glucose or glycerol, preferably glycerol, wherein said inducer,
preferably lactose and said major carbon source, preferably
glycerol are co-fed to said batch culture at a flow rate, wherein
said flow rate is increasing, wherein said flow rate may increase
with a linear or with an exponential characteristic, wherein
preferably the initial flow rate is chosen to to allow feeding of
said major carbon source to said batch culture at about the same
rate as at the end of the growth phase.
[0132] Further alternatively said inducing of said promoter with an
inducer is performed by co-feeding said batch culture with said
inducer and said major carbon source, wherein said inducer is
preferably IPTG or lactose, most preferably lactose, and wherein
said major carbon source is glucose or glycerol, preferably
glycerol, wherein said inducer, preferably lactose is fed to said
batch culture at a first flow rate, and wherein said major carbon
source, preferably glycerol is fed to said batch culture at a
second flow rate, wherein said first flow rate is constant or
increasing, preferably constant, and wherein said second flow rate
is constant or increasing, preferably increasing, wherein
preferably the initial value of said second flow rate is chosen to
to allow feeding of said major carbon source to said batch culture
at about the same rate as at the end of the growth phase. In a very
preferred embodiment said inducer is lactose and said major carbon
source is glycerol, wherein said lactose and said glycerol are
co-fed to said batch culture in a ratio of about 2:1 to 1:4
(w/w).
[0133] The growth of the bacterial host as determined by CDW, CWW
or OD.sub.600 continues during the production phase at a growth
rate which is lower than that during the growth phase and which is
decreasing with the process time. In a further embodiment of the
invention, said inducing said promoter with an inducer is performed
by co-feeding said inducer, preferably lactose and said major
carbon source, preferably glycerol, to said batch culture with an
increasing flow rate, preferably with a flow rate wherein the
incremental increase of the flow rate is adapted to the actual
growth rate of the culture. In a further preferred embodiment said
inducer, preferably lactose, and said major carbon source,
preferably glycerol, are contained in the same medium (co-feed
medium), wherein preferably the ratio between lactose and glycerol
in said medium (co-feed medium) ranges from about 0:1 to 1:0 (w/w),
preferably about 2:1 to about 1:4 (w/w), more preferably about 1:1
to 1:3 (w/w), most preferably the ratio is about 1:3 (w/w). In a
preferred embodiment the ratio of lactose and the major carbon
source, preferably glycerol, is 1:1 (w/w). In another preferred
embodiment the ratio of lactose and the major carbon source,
preferably glycerol, is 1:3 (w/w). In a more preferred embodiment
said medium (co-feed medium) comprises ca. 200 g/l lactose and ca.
200 g/l glycerol. In a still more preferred embodiment the
induction medium comprises ca. 100 g/l lactose and ca. 300 g/l
glycerol.
[0134] In one embodiment of the invention said inducing of said
promoter with an inducer is performed by co-feeding said inducer,
preferably lactose and said major carbon source, preferably
glycerol to said batch culture, wherein said inducer, preferably
lactose and said major carbon source, preferably glycerol are
contained in separate media which are separately fed to said
culture.
[0135] At the end of the production phase the cells are harvested
by centrifugation. Typically, cells are harvested about 5 h after
induction start, when a final OD.sub.600 of 90 to 130 is reached.
Further extension of the production phase leads to higher
OD.sub.600 and CWW values and therefore to further improved yield
of the expression construct.
[0136] Harvested cells may be suspended in a storage buffer and
stored at -80.degree. C. for further processing.
[0137] The total protein content of the cells is determined after
cell lysis by SDS PAGE or LDS PAGE and comparison with a protein
standard. The content of soluble protein is determined by HPLC. The
identity of the expressed capsid protein is determined by western
blotting. The concentration of assembled VLPs can be analysed by
size exclusion chromatography (Example 18). VLP can preparatively
be purified from lysed cells by chromatographic methods.
[0138] Scale-up of the process of the invention to large volumes is
possible with only minor adaptations. The invention encompasses
culture volumes in the range of 100 ml up to 6000 l. Preferred
culture volumes are 40 to 100 l, most preferably about 50 l. It is
apparent for the skilled person that larger culture volumes in
particular require larger volumes of the preculture which is used
for inoculation. For example, a preculture may be performed in two
ore more steps with increasing preculture volume. To ensure plasmid
retention in large culture volumes, the precultures which are used
as inoculum may contain an antibiotic to maintain selection
pressure. The skilled person is aware that plasmid retention can
further be improved by reducing the number of generations which is
necessary to reach the desired final cell density. Therefore, it is
advantageous to inoculate the precultures and the batch cultures
with high cell densities. In a preferred embodiment the initial
OD.sub.600 of the preculture is 0.1 to 0.4, preferably about
0.3.
[0139] In one embodiment, prior to said cultivation step, said
process further comprises the step of introducing said bacterial
host into a medium, wherein said introducing is performed with an
inoculum, wherein said inoculum is produced in a preculture process
comprising the step of growing said bacterial host in a medium
comprising an antibiotic, preferably kanamycin. More preferably,
said preculture process comprises the steps of growing said
bacterial host in a first medium comprising an antibiotic,
preferably kanamycin, and diluting said first medium comprising the
bacterial host with a second medium to an OD.sub.600 of 0.1 to 0.4,
preferably about 0.3, wherein said second medium is essentially
free of an antibiotic, and further cultivating said bacterial
host.
[0140] Furthermore, it is apparent for the skilled person, that the
fermentation process of the invention is an aerobic process which
requires adequate oxygen supply of the bacteria in the culture. The
oxygen demand of the bacterial host is, inter alia, increasing with
increasing cell density and increasing growth rate. Depending on
the total volume and the oxygen demand of the bacterial host,
oxygen can, for example, be supplied by stirring and/or by aeration
with air. Alternatively, oxygen can also be supplied by aeration
with pure oxygen or a mixture of pure oxygen with any other gas,
preferably air, wherein pure oxygen refers to the technically pure
gas as commonly available for technical purposes. A further
possibility of supplying oxygen to the bacterial host is increasing
the oxygen partial pressure in the medium by increasing the
pressure in the fermenter.
[0141] In a preferred embodiment of the invention, said cultivating
said bacterial host and/or said feeding of said batch culture
and/or said inducing of said promoter with an inducer is performed
under conditions, wherein said bacterial host is supplied with
oxygen, preferably by aeration with air, most preferably by
aeration with air in a constant flow, wherein preferably said
oxygen is supplied throughout the entire process, most preferably
throughout the lag-, growth- and production phase, and wherein
further preferably the partial pressure of oxygen is monitored in
the culture medium and wherein the bacterial host is alternatively
or additionally supplied with oxygen by aeration with pure oxygen,
preferably when the partial pressure of oxygen in the medium
(pO.sub.2) is below a certain threshold. In a specifically
preferred embodiment said threshold of pO.sub.2 is in the range of
0% to 60%, preferably 10% to 50%, more preferably 20% to 45% most
preferably said threshold is about 40%.
[0142] Oxygen supply, preferably by aeration with air and/or pure
oxygen to maintain the preferred pO.sub.2 as described above, is
routinely applied in the process of the invention, preferably for
culture volumes of 2 l and more. Aeration with oxygen in the
described manner is especially preferred in the scaled-up process,
most preferably at 40 to 100 l and above. Therefore, one embodiment
of the invention is a process for expression of a recombinant
capsid protein of a bacteriophage or a mutant or fragment thereof
being capable of forming a VLP by self-assembly, said process
comprising the steps of: a) introducing an expression plasmid into
a bacterial host, wherein said expression plasmid comprises an
expression construct, wherein said expression construct comprises
(i) a first nucleotide sequence encoding said recombinant capsid
protein, or mutant or fragment thereof, and (ii) a promoter being
inducible by lactose; b.) cultivating said bacterial host in a
medium comprising a major carbon source; wherein said cultivating
is performed in batch culture and under conditions under which said
promoter is repressed by lad, wherein said lad is overexpressed by
said bacterial host; c.) feeding said batch culture with said major
carbon source; and d.) inducing said promoter with an inducer,
wherein said feeding of said batch culture with said major carbon
source is continued;
[0143] wherein throughout steps b.) to d.) of said process oxygen
is supplied to said bacterial host by a pO.sub.2 in said medium of
at least about 10% to 50%, preferably about 40%, and wherein
further preferably said oxygen is supplied by aeration with air,
pure oxygen, or a mixture of both, preferably by a mixture of air
and pure oxygen.
EXAMPLES
Example 1
Cloning Strategy for the Expression Plasmid pTac-nSD-Qb-mut (SEQ ID
NO:1)
[0144] The coat protein-encoding gene (C) of E. coli RNA
bacteriophage QB is amplified from plasmid pSDQb-mut (SEQ ID
NO:33). The plasmid contains the sequence of gene C coding for the
133-aa Q.beta. coat protein (CP) and the 329-aa read through
protein (A1). To prevent read-through, nucleotides 445-450
according to NCBI GenBank Acc. No. M99030 TGAACA (SEQ ID NO:31) are
replaced by the sequence TAATGA (SEQ ID NO:32).
[0145] The coat protein-encoding gene C from plasmid pSDQb-mut is
amplified by PCR. Oligonucleotide Qb-FOR3/2 (SEQ ID NO:34) with an
internal EcoRI site and a synthetic Shine-Dalgarno (SD, SEQ ID
No:4) sequence anneals to the 5' end of the QB CP gene.
Oligonucleotide Qblang-REV2/2 (SEQ ID NO:35) contains an internal
HindIII site and primes to the 3' end of the noncoding region of
gene C. The 1054 bp amplified PCR fragment includes nucleotides
46-1062 of NCBI GenBank Acc. No. M99039 (except the nucleotide
changes described above) and the synthetic SD sequence. The PCR
fragment is digested with the restriction enzymes HindIIII/EcoRI
and the resulting 1036 bp fragment is inserted into the
HindIII/EcoRI restriction sites of a modified pKK223-3 vector
(Pharmacia, NCBI GenBank Acc. No.: M77749, SEQ ID NO:27). In this
modified pKK223-3 vector the ampicillin resistance gene is replaced
with the kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI
GenBank Acc. No.: X06404, SEQ ID NO:37).
[0146] Vector pTac-nSDQb-mut (SEQ ID NO:33) differs from vector
pTacQb-mut in the Shine-Dalgarno sequence. This Shine-Dalgarno
sequence (nSD, SEQ ID NO:3) is introduced by amplifying the Q.beta.
coat protein-encoding gene C via PCR from plasmid pTacQb-mut.
Oligonucleotide nSDQb-mutEcoRlfor (SEQ ID NO:36) with an internal
EcoRI site and the corresponding synthetic Shine-Dalgarno (nSD)
sequence anneals to the 5' end of the Q.beta. CP gene.
Oligonucleotide Qblang-REV2/2 (SEQ ID NO:35) contains an internal
HindIII site and primes to the 3' end of the noncoding region of
gene C. The 1054 bp amplified PCR fragment includes nucleotides
46-1062 of NCBI GenBank Acc. No. M99039 (except the nucleotide
changes described above) and the synthetic nSD sequence. The PCR
fragment is digested with the restriction enzymes HindIII/EcoRI and
the resulting 1036 bp fragment is inserted into the HindIII/EcoRI
restriction sites of a modified pKK223-3 vector (Pharmacia, NCBI
GenBank Acc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3
vector the ampicillin resistance gene is replaced with the
kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBank
Acc. No.: X06404, SEQ ID NO:37).
Example 2
Cloning Strategy for the Expression Plasmid pTac-nSD-AP205 (SEQ ID
NO:30)
[0147] The coat protein-encoding gene of Acinetobacter
bacteriophage AP205 is amplified from plasmid pAP205-58. This
plasmid contains the sequence of the coat protein gene
(corresponding to nucleotides 1908-2303 of NCBI GenBank Acc. No.
AF334111) coding for the 131-amino acid capsid protein of
bacteriophage AP205.
[0148] The coat protein-encoding gene is amplified by PCR.
Oligonucleotide nSDAP238-EcoRIfor (SEQ ID NO:38) with an internal
EcoRI site and a synthetic Shine-Dalgarno (nSD) sequence anneals to
the 5' end of the coat protein gene. Oligonucleotide
AP238HindIIIrev (SEQ ID NO:39) contains an internal HindIII site
and primes to the 3' end of the coat protein gene. This
oligonucleotid introduces a second stop codon behind the naturally
occurring stop codon of the coat protein. The 438 bp amplified PCR
fragment includes nucleotides 1908-2303 of NCBI GenBank Acc. No.
AF334111 and the synthetic nSD sequence. The PCR fragment is
digested with the restriction enzymes HindIII/EcoRI and the
resulting 420 bp fragment is inserted into the HindIII/EcoRI
restriction sites of a modified pKK223-3 vector (Pharmacia, NCBI
GenBank Acc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3
vector the ampicillin resistance gene is replaced with the
kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBank
Acc. No.: X06404, SEQ ID NO:37).
Example 3
Expression of Q.beta. CP Under Control of the tac Promoter and
nSD
[0149] The E. coli strain RB791 was transformed with plasmids
pTac-nSD-Qb-mut (SEQ ID NO:1). The clone was grown in shake flasks.
Each flask contained 100 ml of R40 medium (main culture medium,
Hypep 7455, glycerol, see Example 5) with kanamycin (25 .mu.g/ml)
and was inoculated with over night cultures at a start OD.sub.600
of 0.3. The shake flasks were incubated for 4 h (OD.sub.600 between
4 and 5) at 30.degree. C. and an agitation of 220 rpm. The
induction was carried out with 0.5% of lactose for 4 h. Protein
production was determined by SDS-PAGE. The gel showed a strong
protein band which was identified as Q.beta. CP.
Example 4
Expression of AP205 CP Under Control of the tac Promoter and SD vs.
nSD
[0150] 9 clones of pTac-nSDAP205 (SEQ ID NO:30) and 6 clones of
pTac-SDAP205 were screened in shake flasks. pTac-SDAP205 (SEQ ID
NO:40) is identical to pTac-nSDAP205 but comprises the
Shine-Dalgarno sequence of SEQ ID NO:4 instead of that of SEQ ID
NO:3. Each flask contained 50 ml of R40 medium (main culture
medium, Hypep 7455, glycerol, see Example 5) with kanamycin (25
.mu.g/ml) and was inoculated with over night cultures at a start
OD.sub.600 of 0.3 (for pTac-nSDAP205) or 0.4 (pTac-SDAP205). The
shake flasks were incubated for 4 h at 30.degree. C. and an
agitation of 220 rpm. The induction was carried out with 0.5% of
lactose. Protein production was determined by SDS-PAGE. For all
tested clones expression of AP205 CP was significantly stronger
from pTac-nSDAP205 than from pTac-SDAP205.
Example 5
Composition of Culture Media
Culture media were composed as described in Table 1.
TABLE-US-00001 [0151] TABLE 1 Composition of Culture media.
Concentrations in [g/L] Main Feed Induction Main Medium + Medium +
Medium + 20% Main Medium + Hypep + 50% Glycerol + 20% Medium +
Bacto Hypep Glycerol Glycerol Lactose YE + Glycerol R27 R40 R41 R42
R43 Component Na.sub.2HPO.sub.4 2H.sub.2O 2.5 2.5 2.5 2.5 2.5
KH.sub.2PO.sub.4 3 3 3 3 3 K.sub.2HPO.sub.4 5.2 5.2 5.2 5.2 5.2
Citrate 3.86 3.86 3.86 3.86 3.86 (NH.sub.4)2SO.sub.4 4 4 4 4 4 Vit
B1 0.01 0.01 0.02 0.02 0.01 CaCl.sub.2 2H.sub.2O 0.0147 0.0147
0.0147 0.0147 0.0147 MgSO.sub.4 7H.sub.2O 0.5 0.5 9 9 0.5
FeCl.sub.3 6H.sub.2O 0.054 0.054 0.054 0.054 0.054 CoCl.sub.2
6H.sub.2O 0.0005 0.0005 0.0005 0.0005 0.0005 MnCl.sub.2 4H.sub.2O
0.003 0.003 0.003 0.003 0.003 CuCl.sub.2 2H.sub.2O 0.0003 0.0003
0.0003 0.0003 0.0003 H.sub.3BO.sub.3 0.003 0.003 0.003 0.003 0.003
Na.sub.2MoO.sub.4 2H.sub.2O 0.0005 0.0005 0.0005 0.0005 0.0005
Zn(CH.sub.3COO).sub.2 2H.sub.2O 0.0026 0.0026 0.0026 0.0026 0.0026
Glucose 5 -- -- -- -- Glycerol -- 5 500 200 5 Lactose anhydrous --
-- -- 200 -- HyPep 7455 5 5 -- -- -- Bacto Yeast Extract -- -- --
-- 5
Example 6
Expression of Q.beta. CP in a Fed-batch Process (2 L Scale)
[0152] The fermentation process was performed in a bioreactor
(Applikon 5 L dished bottom) equipped with 2 disc stirrer (O6 cm),
baffles (3.times.16 cm), pH-, pO2-, and temperature control, and
fermenter software BioXpert Version 2.22
[0153] 5 .mu.L cryo culture of RB791 transformed with plasmids
pTac-nSD-Qb-mut were inoculated in 100 mL Erlenmeyer flasks
containing 50 mL medium R40 (25 .mu.g/mL kanamycin) and cultivated
for 14 h at 30.degree. C. and 220 RPM over night. After 14 h an
OD.sub.600 value of 6.0 was reached. For batch fermentation, 2 L of
medium (R40) were pumped into the bioreactor. In Table 2 the
cultivation parameters are listed.
TABLE-US-00002 TABLE 2 Parameter set points for batch phase.
Parameter Set point Unit Stirrer speed 1000 [rpm] Air supply 2.5
[L/min] O2-supply, maximal 2 [L/min] Temperature 30 [.degree. C.]
O2-saturation >40 [%] pH 6.8 [--]
[0154] The bioreactor was inoculated with 100 mL inoculum. Samples
of 2 mL were taken, OD.sub.600 determined and centrifuged at 14'000
RPM. Pellet and supernatant were separated and frozen for further
analysis. The biomass concentration [g/L] was calculated using the
following equation:
OD.sub.600.times.0.45
[g.times.L.sup.-1.times.OD.sub.600.sup.-1]=biomass [g/L].
[0155] The Qbeta content in percent of the total protein content
was calculated as follows, assuming, that 50% of the E. coli
biomass is protein:
Biomass [g.times.L.sup.-1]/2=total protein [g.times.L.sup.-1]
Qbeta [g.times.L.sup.-1]/total protein
[g.times.L.sup.-1].times.100=Qbeta/total protein [%].
[0156] In the fed-batch mode, which followed the batch mode, a
feeding phase was added. In the feeding phase substrate is supplied
to the cells in the reactor according to a defined profile. The
feed profile depends on the selected growth rate .mu.., the yield
coefficient biomass to glycerol (Y.sub.x/glycerol), the volume
(Vf), and the concentration of substrate in the feed (cf).
substrate concentration. The feed was calculated using the
following equation:
[0157] Feed equation
mf=(.mu./Y.sub.x/s+m) Vf.times.Xf.times.e.sup..mu.t
pump=(mf/cf+b)/a
[0158] mf=mass flow [g/h]
[0159] .mu.=specific growth rate [l/h]
[0160] Y.sub.x/Glycerol=Yield biomass to glycerol [g/g]
[0161] m=maintenance energy [gg.sup.-1h.sup.-1]
[0162] Vf=Volume at feed start
[0163] Xf=Biomass at feed start
[0164] cf=Concentration of substrate in feed [g/mL]
[0165] a+b=offset/slope of pump calibration equation
[0166] For the determination of the calibration parameters a and b,
a pump calibration was carried out. In addition, the feed tube with
feed bottle was clamped into the feed pump and the pump was run
with 7, 14 and 21% pump performance. The pumped feed volume per
time was noted. In a resulting diagram of the relation of pump
performance [%] to pumped feed solution [mL/h], the slope (a) and
the Y-axis section (b) was determined. On the bioreactor the
parameters in Table 3 were set for fed-batch cultivation.
TABLE-US-00003 TABLE 3 Parameters for fed-batch cultivation in
bioreactor. Parameter Set point Unit Stirrer speed 1000 [rpm] Air
supply 2.5 [L/min] O.sub.2-supply, maximal 2 [L/min] Temperature 30
[.degree. C.] O.sub.2-saturation >40 [%] pH 6.8 [--]
[0167] After reaching a process time of approximately 7 h (end of
batch) the feed pump was turned on automatically. After further 7 h
cultivation, when the M.sub.oo reached 55-60, the feed medium (for
biomass propagation) was exchanged with the induction medium R42
(for biomass propagation and induction). After 5 h feeding of R42
was stopped and the culture was harvested by centrifugation.
Analysis of Process Parameters
[0168] The following process parameters were routinely analysed.
The pO.sub.2, pH, temperature and stirrer speed were measured
online throughout the process time. The optical density was
measured offline at 600 nm. The determination of the
.beta.-galactosidase activity was performed using a .beta.-Gal
Assay Kit (Invitrogen, cat. no. K1455-01). The activity was
specified as units per mL OD.sub.600=1.0. It is defined as the
quantity of Ortho-Nitrophenyl-.beta.-D-Galactopyranosid (ONPG) in
nmol, which is hydrolysed per minute and mL bacteria suspension
(OD.sub.600=1.0). The accumulated product was analysed by SDS-PAGE,
the total protein content (soluble and insoluble protein) was
determined and using HPLC analysis, the soluble fraction was
measured. Cell disruption of E. coli was performed in lysis buffer
(50 mM glucose, 25 mM tris/HCl (pH 8), 15 mM EDTA (pH 8.0)) with
and ultrasonic homogeniser (Bandlin Sonoplus, HD2070). 250 .mu.L
bacteria suspension with an OD.sub.600 of 50 were centrifuged with
14000 RPM for 10 min. The pellet was resuspended in 250 .mu.L lysis
buffer (vortex) and placed at room temperature for 5 min
Afterwards, the cells were disrupted for 20 s with ultrasonic at
10% device performance (cells on ice) and then the cell suspension
was centrifuged at 14000 RPM, 10 min The supernatant (soluble
protein) was then analysed by SDS-PAGE and HPLC.
[0169] Samples before induction and at end of production (after 5h
induction) were taken from the bioreactor for analysis of Q.beta.
formation analyzed by SDS-PAGE standardized to OD 5.0. At the end
of cultivation, 1.9 l of the culture was harvested. After
centrifugation, the following cell pellets were obtained in three
independent reactor runs: 1.) End OD.sub.600 of 84: 194 g CWW; 2.)
End OD.sub.600 of 88: 200 g CWW; 3.) End OD.sub.600 of 86: 201 g
CWW.
[0170] The plasmid retention in run 1 and 2 was 100% at induction
start and 100% at harvest. Based on comparison with a Q.beta. CP
standard on SDS-PAGE the yield was roughly estimated to be about 5
g/l Q.beta. CP. HPLC analysis revealed a concentration of about 6
g/l Q.beta. VLP.
Example 7
Selection of Carbon Source and Bacterial Strain
[0171] Glucose and glycerol as carbon sources were compared. In
order to test the growth behaviour of each of the strains DH20 and
RB791 on these carbon sources, shake flask experiments were
conducted with medium containing glucose (R27) and medium
containing glycerol (R40). Both media were supplemented with 25
.mu.g/ml kanamycin. Each culture was started with an initial
OD.sub.600 of 0.3. Induction was performed by adding 0.5% lactose.
The maximum specific growth rates (.mu..sub.max) and the yield
coefficients (Y.sub.x/s) were determined and are listed in Table 4.
RB791 grew faster on both, glucose and glycerol. In addition, the
resulting yield coefficients were higher. Although glucose allowed
higher maximum specific growth rates (.mu..sub.max) the yield
coefficients (Y.sub.x/s) was higher for glycerol.
TABLE-US-00004 TABLE 4 Maximum specific growth rates and the yield
coefficients of the cultivation experiments with RB791 and DH20 on
glucose and glycerol. Yield Max. coefficient spec. (Y.sub.x/s)
growth biomass Value after 4.5 h Culture Time rate from Carbon
Acetate (.mu..sub.max) substrate Strain source OD.sub.600
[g1.sup.-1] [h.sup.-1] [g/g] RB791 glucose 6.24 0.44 0.71 0.72
glycerol 4.04 0.21 0.62 0.86 DH20 glucose 2.52 0.42 0.51 0.71
glycerol 2.82 0.25 0.50 0.81
Example 8
Determination of Optimal Temperature
[0172] The influence of temperature on product formation was
investigated. Two shake flask cultures were inoculated and
incubated at 30.degree. C. and 220 rpm. After an OD.sub.600 of 5
was reached, the cultures were induced with lactose. Subsequently,
one culture was continued to be incubated at 37.degree. C. and the
other culture at 23.degree. C. Results of the SDS-PAGE revealed
that expression levels at 4 and 5 h after induction are higher in
the culture induced at 37.degree. C. Induction of the cultures for
19 h showed a higher Q.beta. level in the cultures induced at
23.degree. C.
Example 9
Induction by Co-Feed of Lactose and Glycerol
[0173] A feed solution of 20% glycerol and 20% lactose was composed
(R42) and applied to fermentation as described in Example 6 at
induction start. FIG. 1 provides an overview over relevant process
parameters throughout the entire process time. Expression was
induced at 13.5 h at an OD.sub.600 of about 55. Upon induction, the
feed pump rate was set to constant. Glycerol did not accumulate
with feeding. Lactose accumulated to 4 g/l and then it started to
diminish. The .beta.-galactosidase activity rose to 10 U/ml and
decreased thereafter. Compared with the previous fermentation runs
a.) lactose applied as a single lactose pulse at induction start,
no feeding; b.) continuous lactose feed without glycerol, the
activity was with 7 U/ml higher and the maximum activity was
already reached after 2 h as compared to 4 h in runs a.) and
b.).
Example 10
Plasmid Retention
[0174] The effect of the following operating conditions on the
plasmid retention was tested in the process described in Example 6:
1.) Preculture starting volume, 2.) Kanamycin in the preculture,
3.) Growth and/or induction at 37.degree. C. vs. 30.degree. C. The
results are summarised in Table 5. Precultures were started with
volumes of 5 .mu.l out of the cell bank vial. Inoculation of a
small volume allowed growth of a preculture over-night. The
preculture for QT0103_F8 contained 25 mg/l kanamycin, whereas the
preculture for QT0103_F7 did not contain any kanamycin. Both
fermentations were operated at 30.degree. C. and induced for 5 h.
Judging from the plasmid retentions before and after 5 h induction,
supplementing the preculture with kanamycin has a positive effect
on plasmid retention. Plasmid retention remained at 98% before and
after 5 h induction. In contrast, plasmid retentions reached only
values of 80% when kanamycin was omitted from the preculture. For a
subsequent run, QT0203_F7, the preculture was also started with 5
.mu.l and grown in kanamycin containing medium. The resulting
fermentation in the bioreactor was operated at 37.degree. C. from
the beginning. Operation at 37.degree. C. had a detrimental effect
on the plasmid stability. While the plasmid retention was at 99%
before induction, it dropped to 0% after 5 h induction. In order to
test whether a shorter preculture and thus, less generations, would
improve the plasmid retention after 5 h induction, a set of
precultures were started with 300 .mu.l volume from a thawed cell
bank vial and grown in kanamycin free medium. Two fermenters were
operated at 30.degree. C. for the whole run. An additional two
fermenters were operated first at 30.degree. C. for cell growth and
than switched to 37.degree. C. for the production phase. The
resulting plasmid stabilities were all at 100% before and 5 h after
induction.
TABLE-US-00005 TABLE 5 Summary of plasmid retention before and 5 h
after induction obtained under different operating conditions in
terms of generations in the preculture, with and without kanamycin
in the preculture, and growth and/or induction at 37.degree. C.
Plasmid Plasmid Preculture Kana- retention retention Starting mycin
in before after 5 h Bioreactor Culture pre- induc. induc. run
Volume culture [%] [h] Remarks QT0103_F8 5 25 mg/L 98 98 whole
process at 30.degree. C. QT0103_F7 5 no 80 80 whole process at
30.degree. C. QT0203_F7 5 25 mg/L 99 0 Bioreactor run at 37.degree.
C. QT0603_F7 300 no 100 100 whole process at 30.degree. C.
QT0703_F8 300 no 100 100 Induction at 37.degree. C., rest of the
process at 30.degree. C. QT0803_F9 300 no 100 100 whole process at
30.degree. C. QT0903_F10 300 no 100 100 Induction at 37.degree. C.,
rest of the process at 30.degree. C.
Example 11
Variation in Time Point of Induction
[0175] In a process essentially as described in Example 6 the
exponential feed profile was programmed to start 7 h after the
inoculation of the bioreactor. Under standard conditions, the
scheduled time for induction was at 14 h process time. In order to
test the effect of variations in the time point of induction on the
final cell densities, one culture was induced at 13.5 h (resulting
in 6.5 h of exponential feed) and another culture at 14.5 h
(resulting in 7.5 h of exponential feed). One culture induced at
the regular 14 h time point served as a control (7 h of exponential
feed). Results are summarised in Table 6. Cell density increased
with increasing length of feeding.
[0176] Judged from a linear regression analysis of the available
data points for final CWW, a linear relationship appears to exist
(r.sup.2=0.92).
TABLE-US-00006 TABLE 6 Variations in time point of induction:
effect on final cell density in terms of OD.sub.600 and CWW.
Process Time Duration of Final Point of Exp. Feed Final CWW Reactor
Induction Phase [h] OD.sub.600 [g/L] F2 13 h 32 min 6.5 83.4 116.5
Fl 14 h 02 min 7.0 82.4 122.5 F3 14 h 29 min 7.5 100.4 141.1
Example 12
Variation in Time Point of Harvest
[0177] Harvest of the culture in a process essentially as described
in Example 6 is performed manually. Under standard conditions, the
scheduled time for harvest was at 19 h process time. The operation
"Harvest" involves the manual ending of the bioreactor operations.
In order to test the effect of variations in the time point of
harvest on the final cell densities, one culture was harvested at
18.8 h (resulting in 4.8 h of induction) and another culture at
19.5 h (resulting in 5.5 h of induction). One culture harvested at
the regular 19 h time point served as a control (5 h of induction).
Results are summarized in Table 7. Cell density increased with
increasing length of induction because the cells are still growing
while induced.
TABLE-US-00007 TABLE 7 Variation in time point of harvest: effect
on final cell density in terms of OD600 and CWW. Process Time
Length of Final Point of Induction Final CWW Reactor Harvest [h]
OD.sub.600 [g/L] F5 18 h 50 min 4.8 91.4 122.4 F4 19 h 00 min 5.0
92.2 127.5 F6 19 h 30 min 5.5 96.0 132.4
Example 13
Effect of Temperature
[0178] The effect of fermentation temperature in a process
essentially as described in Example 6 was investigated by running 6
fermentations at 5 different temperature setpoints. Results are
summarized in Table 8. Final cell densities were sensitive to the
fermentation temperature with an optimum at a temperature of
30.degree. C.
TABLE-US-00008 TABLE 8 Summarized results of different temperature
setpoints on final cell density in terms of 0D600 and CWW.
Temperature Final CWW Reactor [.degree. C.] Final OD.sub.600 [g/L]
F5 25.0 37.8 62 F4 27.5 80.0 117 F3 30.0 92.8 123 F4 30.0 92.4 125
F5 32.5 85.0 111 F6 35.0 79.6 107
Example 14
Scaled-Up Fermentation (50 1)
[0179] The process described in Example 6 was scaled up to a volume
of 50 l order to evaluate scale-up capability from the 2 L working
volume bioreactor system to a larger volume. Key process parameters
for the scaled-up process are summarized in Table 9.
TABLE-US-00009 TABLE 9 Process parameters of in 50 L bioreactor.
Time Culture Step Description [h] OD.sub.600 Preculture 1 300 .mu.l
from cell bank vial are transferred -11* 5.0 into 100 ml preculture
medium contained in 500 mL shake flask and cultured for 16 h
Preculture 2 Calculate the required volume for -5* 4.0 transfer in
order to start with initial OD.sub.600 of 0.3 in 750 ml. Tranfer
calculated volume (e.g. 50 ml) into 750 ml preculture medium
contained in 5000 mL shake flask Inoculation Pooled calculated
volume (e.g. 1.4 L) is 0 of Bioreactor transferred into the 50 L
Bioreactor. Initial volume: =40 L Induction The exponential feeding
profile is 14 46 Start switched to constant and feed is switched to
induction feed End of Culture is completed after 5 h 19 128 Culture
of induction *Relative to the time of bioreactor inoculation.
[0180] It was necessary to have two preculture expansion steps. In
the first step, the cells were expanded as established for the 2 L
process (Example 6). After this step, cells were split into two
5000 ml shake flask cultures, containing 750 ml medium each.
Further expansion was performed for 5 h. The cultures in the 50 L
bioreactors were performed with the same time profile as in the 2 L
system (Example 6). OD.sub.600 at induction start was 46, the final
OD.sub.600 was 128. Plasmid retention was 100% before induction and
98% at the end of culture. The concentration of Q.beta. CP protein
in the medium at the end of culture was roughly estimated 8 g/l
using SDS-PAGE. The total amount of Q.beta. was estimated about 300
g for this reactor run.
Example 15
Effect of Extended Exponential Feed
[0181] The exponential feeding phase for fermentations performed
according to Examples 6 or 14 was 7 h. After this time the cells
reached a density for induction, which increased during induction
to the targeted maximum 0D.sub.600 of around 100 to 130 as final
cell density. Final OD.sub.600, final CWW, final CDW, plasmid
retention at induction start and harvest and Q.beta. concentration
at the end of culture are determined for reactor runs performed as
described in Examples 6 and 14, preferably as in Example 14,
wherein the exponential feeding phase is extended to a duration up
to 11 h, preferably to 10 h.
Example 16
Effect of Increased Feeding During Production
[0182] Example 9 demonstrates that the glycerol does not accumulate
during production phase, indicating that production might be
limited by the feeding rate of induction medium. Effect of extended
feeding rate of induction medium on final OD.sub.600, final CWW,
final CDW, plasmid retention at induction start and harvest and
Q.beta. concentration at the end of culture is determined in
reactor runs as described in Example 6 and 14, preferably as in
Example 14, wherein the feeding rate during production is
increased. Alternatively or additionally, the ratio between lactose
and glycerol in the feed medium shifted towards a higher glycerol
and a lower lactose concentration.
Example 17
HPLC Analysis of Q.beta. CP
[0183] Q.beta. CP was measured with an HPLC system as follows: A
sample containing Q.beta. CP was diluted appropriately in 1.times.
reaction buffer (50 mM tris(hydroxymethyl)aminomethane buffer pH
8.0) containing 10 mM 1,4-Dithio-DL-threitol and incubated for 15
min at 50.degree. C. in a thermomixer. After incubation the sample
was centrifuged and the supernatant was stored at 2.degree. C. to
10.degree. C. until HPLC analysis. 10 to 100 .mu.l of the sample
were injected.
[0184] Q.beta. was quantified with a regression curve of known
Q.beta. standards regressed to the HPLC peak area detected at 215
nm after elution from a C.sub.4 reversed phase column, 300 .ANG., 5
.mu.m, 4.6.times.150 mm, Vydac Inc., Hesperia, USA (Cat. No.
214TP5415) thermally equilibrated at 50.degree. C. The flow rate
through the system was 1 ml/min consisting of mobile phase A (0.12%
trifluoroacetic acid in water) and mobile phase B (0.12%
trifluoroacetic acid in acetonitrile) with the following gradient
of phase B: 0 to 2 min constant at 40%, 2 to 8 min linear increase
to 50%, 8 to 10 min constant at 50%, 10 to 10.1 min linear decrease
to 40%, and 10.1 to 12 min constant at 40%.
Example 18
Determination of Q.beta. VLP by Analytical Size Exclusion
Chromatography
[0185] Analysis of Q.beta. particles by analytical size exclusion
chromatography was performed using a Tskge1G5000 PW.sub.XL-column
(10 .mu.m, 7.8.times.300 mm, TosoH Biosep; Cat.-No. 08023)
equilibrated in phosphate buffered saline (20 mM
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, 150 mM NaCl pH 7.2). Elution
was performed by an isocratic gradient for 20 min at 0.8 ml/min in
phosphate buffered saline. The Qbeta concentration was determined
from a regression curve of known Q.beta. standards regressed to the
HPLC peak area detected at 260 nm.
Example 19
Effect of Extended Exponential Feed
[0186] The exponential feeding phase for fermentations performed
according to Examples 6 or 14 was 7 h. After this time the cells
reached a density for induction, which increased during induction
to the targeted maximum OD.sub.600 of around 100 to 130 as final
cell density. Final OD.sub.600, final CWW, plasmid retention before
induction and at harvest and Q.beta. concentration at the end of
culture were determined for reactor runs performed as described in
Examples 6 and 14, preferably as in Example 14, wherein the
exponential feeding phase was extended to a duration up to 12 h. In
addition, the concentration of glycerol and lactose in the
induction feed were changed to 300 g/L and 100 g/L respectively.
The results are summarized in Table 10.
TABLE-US-00010 TABLE 10 OD.sub.600 and CWW at the end of
cultivations, Plasmid Retention before induction and at the end of
cultivations as well as the peak oxygen mass flow. The cultivations
were conducted with different duration of exponential feeding.
Plasmid Retention Duration [%] Peak Oxygen Exponential OD.sub.600
CWW before end of Mass Flow Feeding [h] [--] [g/L] induction
cultivation [vvm] 7 86 122 100 99 0.2 8 112 184 99 98 0.4 9 136 217
100 98 0.8 10 164 228 99 98 1.5 11 200 262 100 97 >4.5 12 90 186
99 100 >4.5
[0187] According to LDS-PAGE analysis, the specific Qbeta
concentration of all cultivations except for the cultivation with
12 h exponential feeding was the same. An optimum regarding
absolute Qbeta yield and oxygen consumption was found for 9.5 h
exponential feeding. Therefore, the process is preferably run with
9.5 h exponential feeding phase.
Example 20
Scaled Up Fermentation (50 l)
[0188] The process described in Example 6 and with 9.5 h
exponential feeding phase with 300 g/L glycerol and 100 g/L lactose
as described in Example 19 was scaled up to a volume of 50 L in
order to evaluate scale-up capability from the 2 L working volume
bioreactor system to a larger volume. Key process parameters for
the scaled up process are summarized in Table 11.
TABLE-US-00011 TABLE 11 Process parameters on the 50 L scale Time
Culture Step Description [h] Preculture 200 .mu.l from cell bank
vial were transferred -18* into 800 ml preculture medium contained
in 3000 mL shake flask and cultured for 18 h (2 flasks) Inoculation
Pooled total volume (approx. 1.6 L) was 0 of Bioreactor transferred
into the 50 L Bioreactor. Initial volume: =35 L Induction The
exponential feeding profile was 16.5 Start switched to constant and
feed was switched to induction feed End of Culture was completed
after 5 h of 21.5 Culture induction *Relative to the time of
bioreactor inoculation.
[0189] It was necessary to change the preculture procedure in order
to inoculate the larger reactor with approximately the same cell
density. The cultures in the 50 L bioreactors were performed with
the time profile optimised for the 2 L scale as described in
Example 19. The final cell wet weight for six cultivations was 188
g/L.+-.9. Plasmid retention was 97.3%.+-.1.4 at the end of culture.
The concentration of Q.beta. CP protein in the medium at the end of
culture was determined by C.sub.4 reversed phase HPLC (Example 17)
to 10.8 g/L.+-.0.3. The total amount of Q.beta. CP was 540 g for
one 50 L run. The crude extract of approximately two times
concentrated biomass was analysed for Q.beta. CP and Q.beta. VLP
(Example 18). The concentration of Q.beta. CP was 19.1 g/L.+-.0.4
(C.sub.4 reversed phase HPLC), the concentration of Q.beta. VLP was
18.8 g/L.+-.1.1. Therefore, the VLP-yield of the fermentation
process is estimated to approximately 9-11 g/l fermentation broth
at the time of harvest.
Sequence CWU 1
1
4015579DNAartificial sequenceplasmid 1ggctgtgcag gtcgtaaatc
actgcataat tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga
catcataacg gttctggcaa atattctgaa atgagctgtt 120gacaattaat
catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
180ggaaacagaa ttctaaggag gaaaaaaaaa tggcaaaatt agagactgtt
actttaggta 240acatcgggaa agatggaaaa caaactctgg tcctcaatcc
gcgtggggta aatcccacta 300acggcgttgc ctcgctttca caagcgggtg
cagttcctgc gctggagaag cgtgttaccg 360tttcggtatc tcagccttct
cgcaatcgta agaactacaa ggtccaggtt aagatccaga 420acccgaccgc
ttgcactgca aacggttctt gtgacccatc cgttactcgc caggcatatg
480ctgacgtgac cttttcgttc acgcagtata gtaccgatga ggaacgagct
tttgttcgta 540cagagcttgc tgctctgctc gctagtcctc tgctgatcga
tgctattgat cagctgaacc 600cagcgtatta atgactgctc attgccggtg
gtggctcagg gtcaaaaccc gatccggtta 660ttccggatcc accgattgat
ccgccgccag ggacaggtaa gtatacctgt cccttcgcaa 720tttggtccct
agaggaggtt tacgagcctc ctactaagaa ccgaccgtgg cctatctata
780atgctgttga actccagcct cgcgaatttg atgttgccct caaagatctt
ttgggcaata 840caaagtggcg tgattgggat tctcggctta gttataccac
gttccgcggt tgccgtggca 900atggttatat tgaccttgat gcgacttatc
ttgctactga tcaggctatg cgtgatcaga 960agtatgatat tcgcgagggc
aagaaacctg gtgctttcgg taacattgag cgattcattt 1020atcttaagtc
gataaatgct tattgctctc ttagcgatat tgcggcctat cacgccgatg
1080gcgtgatagt tggcttttgg cgcgatccat ccagtggtgg tgccataccg
tttgacttca 1140ctaagtttga taagactaaa tgtcctattc aagccgtgat
agtcgttcct cgtgcttagt 1200aactaaggat gaaatgcatg tctaagcttg
gctgttttgg cggatgagag aagattttca 1260gcctgataca gattaaatca
gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg 1320gcagtagcgc
ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
1380ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
tcaaataaaa 1440cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
gttgtttgtc ggtgaacgct 1500ctcctgagta ggacaaatcc gccgggagcg
gatttgaacg ttgcgaagca acggcccgga 1560gggtggcggg caggacgccc
gccataaact gccaggcatc aaattaagca gaaggccatc 1620ctgacggatg
gcctttttgc gtttctacaa actcttttgt ttatttttct agagccacgt
1680tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
catgaacaat 1740aaaactgtct gcttacataa acagtaatac aaggagtgtt
atgagccata ttcaacggga 1800aacgtcttgc tcgaggccgc gattaaattc
caacatggat gctgatttat atgggtataa 1860atgggctcgc gataatgtcg
ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 1920cgatgcgcca
gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
1980tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
tcaagcattt 2040tatccgtact cctgatgatg catggttact caccactgcg
atccccggga aaacagcatt 2100ccaggtatta gaagaatatc ctgattcagg
tgaaaatatt gttgatgcgc tggcagtgtt 2160cctgcgccgg ttgcattcga
ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 2220tcgtctcgct
caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
2280tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
agcttttgcc 2340attctcaccg gattcagtcg tcactcatgg tgatttctca
cttgataacc ttatttttga 2400cgaggggaaa ttaataggtt gtattgatgt
tggacgagtc ggaatcgcag accgatacca 2460ggatcttgcc atcctatgga
actgcctcgg tgagttttct ccttcattac agaaacggct 2520ttttcaaaaa
tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
2580cgatgagttt ttctaaacgc gtgaccaagt ttactcatat gtactttaga
ttgatttaaa 2640acttcatttt taatttaaaa ggatctaggt gaagatcctt
tttgataatc tcatgaccaa 2700aatcccttaa cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa agatcaaagg 2760atcttcttga gatccttttt
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 2820gctaccagcg
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
2880tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
agttaggcca 2940ccacttcaag aactctgtag caccgcctac atacctcgct
ctgctaatcc tgttaccagt 3000ggctgctgcc agtggcgata agtcgtgtct
taccgggttg gactcaagac gatagttacc 3060ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc acacagccca gcttggagcg 3120aacgacctac
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
3180cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
gagagcgcac 3240gagggagctc ccagggggaa acgcctggta tctttatagt
cctgtcgggt ttcgccacct 3300ctgacttgag cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat ggaaaaacgc 3360cagcaacgcg gcctttttac
ggttcctggc cttttgctgg ccttttgctc acatgttctt 3420tcctgcgtta
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
3480cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
cggaagagcg 3540cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
tcacaccgca tatggtgcac 3600tctcagtaca atctgctctg atgccgcata
gttaagccag tatacactcc gctatcgcta 3660cgtgactggg tcatggctgc
gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3720gcttgtctgc
tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
3780tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
aagctcatca 3840gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
ccgcgtccag ctcgttgagt 3900ttctccagaa gcgttaatgt ctggcttctg
ataaagcggg ccatgttaag ggcggttttt 3960tcctgtttgg tcactgatgc
ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 4020ccgatgaaac
gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
4080ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
gagaaaaatc 4140actcagggtc aatgccagcg cttcgttaat acagatgtag
gtgttccaca gggtagccag 4200cagcatcctg cgatgcagat ccggaacata
atggtgcagg gcgctgactt ccgcgtttcc 4260agactttacg aaacacggaa
accgaagacc attcatgttg ttgctcaggt cgcagacgtt 4320ttgcagcagc
agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
4380aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
gcgcacccgt 4440ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
tgctggagat ggcggacgcg 4500atggatatgt tctgccaagg gttggtttgc
gcattcacag ttctccgcaa gaattgattg 4560gctccaattc ttggagtggt
gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg 4620aggtggcccg
gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
4680cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
atcgccgtga 4740cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
cgcgagcgat ccttgaagct 4800gtccctgatg gtcgtcatct acctgcctgg
acagcatggc ctgcaacgcg ggcatcccga 4860tgccgccgga agcgagaaga
atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg 4920ccagcaagac
gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
4980cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
aagattccga 5040ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
aaagcggtcc tcgccgaaaa 5100tgacccagag cgctgccggc acctgtccta
cgagttgcat gataaagaag acagtcataa 5160gtgcggcgac gatagtcatg
ccccgcgccc accggaagga gctgactggg ttgaaggctc 5220tcaagggcat
cggtcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
5280gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
gagatggcgc 5340ccaacagtcc cccggccacg gggcctgcca ccatacccac
gccgaaacaa gcgctcatga 5400gcccgaagtg gcgagcccga tcttccccat
cggtgatgtc ggcgatatag gcgccagcaa 5460ccgcacctgt ggcgccggtg
atgccggcca cgatgcgtcc ggcgtagagg atccgggctt 5520atcgactgca
cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtat
55792245DNAartificial sequencepromoter sequence 2cgactgcacg
gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc 60tgtgcaggtc
gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat
120aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg
agctgttgac 180aattaatcat cggctcgtat aatgtgtgga attgtgagcg
gataacaatt tcacacagga 240aacag 245319DNAartificial
sequenceShine-Dalgarno Sequence 3taaggaggaa aaaaaaatg
19418DNAartificial sequenceShine-Dalgarno Sequence 4aggaggtaaa
aaacgatg 185133PRTBacteriophage Qbeta 5Met Ala Lys Leu Glu Thr Val
Thr Leu Gly Asn Ile Gly Lys Asp Gly1 5 10 15Lys Gln Thr Leu Val Leu
Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ser Leu Ser
Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val
Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser65 70 75 80Cys
Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser 85 90
95Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu
100 105 110Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile
Asp Gln 115 120 125Leu Asn Pro Ala Tyr 13061017DNAartificial
sequenceExpression construct 6atggcaaaat tagagactgt tactttaggt
aacatcggga aagatggaaa acaaactctg 60gtcctcaatc cgcgtggggt aaatcccact
aacggcgttg cctcgctttc acaagcgggt 120gcagttcctg cgctggagaa
gcgtgttacc gtttcggtat ctcagccttc tcgcaatcgt 180aagaactaca
aggtccaggt taagatccag aacccgaccg cttgcactgc aaacggttct
240tgtgacccat ccgttactcg ccaggcatat gctgacgtga ccttttcgtt
cacgcagtat 300agtaccgatg aggaacgagc ttttgttcgt acagagcttg
ctgctctgct cgctagtcct 360ctgctgatcg atgctattga tcagctgaac
ccagcgtatt aatgactgct cattgccggt 420ggtggctcag ggtcaaaacc
cgatccggtt attccggatc caccgattga tccgccgcca 480gggacaggta
agtatacctg tcccttcgca atttggtccc tagaggaggt ttacgagcct
540cctactaaga accgaccgtg gcctatctat aatgctgttg aactccagcc
tcgcgaattt 600gatgttgccc tcaaagatct tttgggcaat acaaagtggc
gtgattggga ttctcggctt 660agttatacca cgttccgcgg ttgccgtggc
aatggttata ttgaccttga tgcgacttat 720cttgctactg atcaggctat
gcgtgatcag aagtatgata ttcgcgaggg caagaaacct 780ggtgctttcg
gtaacattga gcgattcatt tatcttaagt cgataaatgc ttattgctct
840cttagcgata ttgcggccta tcacgccgat ggcgtgatag ttggcttttg
gcgcgatcca 900tccagtggtg gtgccatacc gtttgacttc actaagtttg
ataagactaa atgtcctatt 960caagccgtga tagtcgttcc tcgtgcttag
taactaagga tgaaatgcat gtctaag 10177132PRTBacteriophage Qbeta 7Ala
Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
1308132PRTBacteriophage Qbeta 8Ala Lys Leu Glu Thr Val Thr Leu Gly
Lys Ile Gly Lys Asp Gly Lys1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg
Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly
Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln
Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln
Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser
Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln
Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105
110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu
115 120 125Asn Pro Ala Tyr 1309132PRTBacteriophage Qb 9Ala Arg Leu
Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10 15Gln Thr
Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala
Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40
45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val
50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser
Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr
Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val
Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile
Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13010132PRTBacteriophage Qbeta 10Ala Lys Leu Glu Thr Val Thr Leu
Gly Asn Ile Gly Lys Asp Gly Arg1 5 10 15Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala
Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser
Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile
Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro
Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr
Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105
110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu
115 120 125Asn Pro Ala Tyr 13011132PRTBacteriophage Qbeta 11Ala Arg
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg1 5 10 15Gln
Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25
30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val
35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys
Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly
Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val
Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe
Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu
Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13012131PRTBacteriophage AP205 12Met Ala Asn Lys Pro Met Gln Pro
Ile Thr Ser Thr Ala Asn Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg
Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys
Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser
Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala
Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val
Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp
Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105
110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp
115 120 125Thr Thr Ala 13013131PRTBacteriophage AP205 13Met Ala Asn
Lys Thr Met Gln Pro Ile Thr Ser Thr Ala Asn Lys Ile1 5 10 15Val Trp
Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu
Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40
45Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly
50 55 60Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile
Arg65 70 75 80Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu
Lys Ala Glu 85 90 95Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe
Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala
Ala Ile Val Ser Ser Asp 115 120 125Thr Thr Ala
13014131PRTBacteriophage AP205 14Met Ala Asn Lys Pro Met Gln Pro
Ile Thr Ser Thr Ala Asp Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg
Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys
Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser
Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala
Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val
Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp
Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105
110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp
115 120 125Thr Thr Ala 13015329PRTBacteriophage Qbeta 15Met Ala Lys
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly1 5 10 15Lys Gln
Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val
Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40
45Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys
50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly
Ser65
70 75 80Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe
Ser 85 90 95Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg
Thr Glu 100 105 110Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp
Ala Ile Asp Gln 115 120 125Leu Asn Pro Ala Tyr Trp Thr Leu Leu Ile
Ala Gly Gly Gly Ser Gly 130 135 140Ser Lys Pro Asp Pro Val Ile Pro
Asp Pro Pro Ile Asp Pro Pro Pro145 150 155 160Gly Thr Gly Lys Tyr
Thr Cys Pro Phe Ala Ile Trp Ser Leu Glu Glu 165 170 175Val Tyr Glu
Pro Pro Thr Lys Asn Arg Pro Trp Pro Ile Tyr Asn Ala 180 185 190Val
Glu Leu Gln Pro Arg Glu Phe Asp Val Ala Leu Lys Asp Leu Leu 195 200
205Gly Asn Thr Lys Trp Arg Asp Trp Asp Ser Arg Leu Ser Tyr Thr Thr
210 215 220Phe Arg Gly Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp Ala
Thr Tyr225 230 235 240Leu Ala Thr Asp Gln Ala Met Arg Asp Gln Lys
Tyr Asp Ile Arg Glu 245 250 255Gly Lys Lys Pro Gly Ala Phe Gly Asn
Ile Glu Arg Phe Ile Tyr Leu 260 265 270Lys Ser Ile Asn Ala Tyr Cys
Ser Leu Ser Asp Ile Ala Ala Tyr His 275 280 285Ala Asp Gly Val Ile
Val Gly Phe Trp Arg Asp Pro Ser Ser Gly Gly 290 295 300Ala Ile Pro
Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys Pro Ile305 310 315
320Gln Ala Val Ile Val Val Pro Arg Ala 32516129PRTBacteriophage R17
16Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly1
5 10 15Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
Trp 20 25 30Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys
Ser Val 35 40 45Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys
Val Glu Val 50 55 60Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu
Leu Pro Val Ala65 70 75 80Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu
Thr Ile Pro Ile Phe Ala 85 90 95Thr Asn Ser Asp Cys Glu Leu Ile Val
Lys Ala Met Gln Gly Leu Leu 100 105 110Lys Asp Gly Asn Pro Ile Pro
Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120
125Tyr17130PRTBacteriophage fr 17Met Ala Ser Asn Phe Glu Glu Phe
Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val Lys Val Ala Pro
Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile Ser Ser Asn Ser
Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val Arg Gln Ser Ser
Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu 50 55 60Val Pro Lys Val
Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val65 70 75 80Ala Ala
Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro Val Phe 85 90 95Ala
Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala Leu Gln Gly Thr 100 105
110Phe Lys Thr Gly Asn Pro Ile Ala Thr Ala Ile Ala Ala Asn Ser Gly
115 120 125Ile Tyr 13018130PRTBacteriophage GA 18Met Ala Thr Leu
Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly1 5 10 15Asn Val Thr
Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30Leu Ser
Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45Arg
Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu Val 50 55
60Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Gly Ser65
70 75 80Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile Phe
Ala 85 90 95Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu Ala Gly
Leu Phe 100 105 110Lys Val Gly Asn Pro Ile Ala Glu Ala Ile Ser Ser
Gln Ser Gly Phe 115 120 125Tyr Ala 13019132PRTBacteriophage SP
19Met Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly1
5 10 15Asp Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn
Gly 20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu
Lys Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys
Asn Phe Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr
Arg Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Ala Phe Ala
Asp Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Asp Glu Glu Arg
Ala Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Asp Pro
Leu Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr
13020329PRTBacteriophage 20Ala Lys Leu Asn Gln Val Thr Leu Ser Lys
Ile Gly Lys Asn Gly Asp1 5 10 15Gln Thr Leu Thr Leu Thr Pro Arg Gly
Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Glu Ala Gly Ala
Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ala Gln Pro
Ser Arg Asn Arg Lys Asn Phe Lys Val 50 55 60Gln Ile Lys Leu Gln Asn
Pro Thr Ala Cys Thr Arg Asp Ala Cys Asp65 70 75 80Pro Ser Val Thr
Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe Thr 85 90 95Ser Tyr Ser
Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu Ala 100 105 110Ala
Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu Asn 115 120
125Pro Ala Tyr Trp Ala Ala Leu Leu Val Ala Ser Ser Gly Gly Gly Asp
130 135 140Asn Pro Ser Asp Pro Asp Val Pro Val Val Pro Asp Val Lys
Pro Pro145 150 155 160Asp Gly Thr Gly Arg Tyr Lys Cys Pro Phe Ala
Cys Tyr Arg Leu Gly 165 170 175Ser Ile Tyr Glu Val Gly Lys Glu Gly
Ser Pro Asp Ile Tyr Glu Arg 180 185 190Gly Asp Glu Val Ser Val Thr
Phe Asp Tyr Ala Leu Glu Asp Phe Leu 195 200 205Gly Asn Thr Asn Trp
Arg Asn Trp Asp Gln Arg Leu Ser Asp Tyr Asp 210 215 220Ile Ala Asn
Arg Arg Arg Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp225 230 235
240Ala Thr Ala Met Gln Ser Asp Asp Phe Val Leu Ser Gly Arg Tyr Gly
245 250 255Val Arg Lys Val Lys Phe Pro Gly Ala Phe Gly Ser Ile Lys
Tyr Leu 260 265 270Leu Asn Ile Gln Gly Asp Ala Trp Leu Asp Leu Ser
Glu Val Thr Ala 275 280 285Tyr Arg Ser Tyr Gly Met Val Ile Gly Phe
Trp Thr Asp Ser Lys Ser 290 295 300Pro Gln Leu Pro Thr Asp Phe Thr
Gln Phe Asn Ser Ala Asn Cys Pro305 310 315 320Val Gln Thr Val Ile
Ile Ile Pro Ser 32521130PRTBacteriophage MS2 21Met Ala Ser Asn Phe
Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val Thr
Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile Ser
Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60Val
Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val65 70 75
80Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly
Leu 100 105 110Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala
Asn Ser Gly 115 120 125Ile Tyr 13022133PRTBacteriophage M11 22Met
Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Lys Gly1 5 10
15Asp Val Thr Leu Asp Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly
20 25 30Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys
Arg 35 40 45Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala
Ser Gly Thr65 70 75 80Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ser
Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Val Glu Glu Arg
Ala Leu Val Arg Thr Glu 100 105 110Leu Gln Ala Leu Leu Ala Asp Pro
Met Leu Val Asn Ala Ile Asp Asn 115 120 125Leu Asn Pro Ala Tyr
13023133PRTBacteriophage MX1 23Met Ala Lys Leu Gln Ala Ile Thr Leu
Ser Gly Ile Gly Lys Asn Gly1 5 10 15Asp Val Thr Leu Asn Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ala Leu Ser Glu Ala
Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Ile Ser Val Ser
Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile
Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr65 70 75 80Cys Asp Pro
Ser Val Thr Arg Ser Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr
Gln Tyr Ser Thr Asp Glu Glu Arg Ala Leu Val Arg Thr Glu 100 105
110Leu Lys Ala Leu Leu Ala Asp Pro Met Leu Ile Asp Ala Ile Asp Asn
115 120 125Leu Asn Pro Ala Tyr 13024330PRTBacteriophage NL95 24Met
Ala Lys Leu Asn Lys Val Thr Leu Thr Gly Ile Gly Lys Ala Gly1 5 10
15Asn Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly
20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys
Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys
Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp
Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala
Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Lys Asp Asp Leu
Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr Trp Ala
Ala Leu Leu Ala Ala Ser Pro Gly Gly Gly 130 135 140Asn Asn Pro Tyr
Pro Gly Val Pro Asp Ser Pro Asn Val Lys Pro Pro145 150 155 160Gly
Gly Thr Gly Thr Tyr Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly 165 170
175Glu Leu Ile Thr Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys
180 185 190Gly Ser Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp
Phe Leu 195 200 205Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu
Ser Lys Tyr Asp 210 215 220Ile Glu Thr His Arg Arg Cys Arg Gly Asn
Gly Tyr Val Asp Leu Asp225 230 235 240Ala Ser Val Met Gln Ser Asp
Glu Tyr Val Leu Ser Gly Ala Tyr Asp 245 250 255Val Val Lys Met Gln
Pro Pro Gly Thr Phe Asp Ser Pro Arg Tyr Tyr 260 265 270Leu His Leu
Met Asp Gly Ile Tyr Val Asp Leu Ala Glu Val Thr Ala 275 280 285Tyr
Arg Ser Tyr Gly Met Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295
300Pro Gln Leu Pro Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys
Pro305 310 315 320Val Gln Thr Val Ile Val Ile Pro Ser Leu 325
33025129PRTBacteriophage f2 25Ala Ser Asn Phe Thr Gln Phe Val Leu
Val Asn Asp Gly Gly Thr Gly1 5 10 15Asn Val Thr Val Ala Pro Ser Asn
Phe Ala Asn Gly Val Ala Glu Trp 20 25 30Ile Ser Ser Asn Ser Arg Ser
Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45Arg Gln Ser Ser Ala Gln
Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60Pro Lys Val Ala Thr
Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala65 70 75 80Ala Trp Arg
Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105
110Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile
115 120 125Tyr26128PRTBacteriophage PP7 26Met Ser Lys Thr Ile Val
Leu Ser Val Gly Glu Ala Thr Arg Thr Leu1 5 10 15Thr Glu Ile Gln Ser
Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val 20 25 30Gly Pro Leu Val
Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn 35 40 45Gly Ala Lys
Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln Ala Asp 50 55 60Val Val
Asp Cys Ser Thr Ser Val Cys Gly Glu Leu Pro Lys Val Arg65 70 75
80Tyr Thr Gln Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr
85 90 95Glu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val
Ala 100 105 110Thr Ser Gln Val Glu Asp Leu Val Val Asn Leu Val Pro
Leu Gly Arg 115 120 125274586DNAartificial sequenceCloning Vector
27ttctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccgatg
60attaattgtc aacagctcat ttcagaatat ttgccagaac cgttatgatg tcggcgcaaa
120aaacattatc cagaacggga gtgcgccttg agcgacacga attatgcagt
gatttacgac 180ctgcacagcc ataccacagc ttccgatggc tgcctgacgc
cagaagcatt ggtgcaccgt 240gcagtcgata agctccggat cctctacgcc
ggacgcatcg tggccggcat caccggcgcc 300acaggtgcgg ttgctggcgc
ctatatcgcc gacatcaccg atggggaaga tcgggctcgc 360cacttcgggc
tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
420ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
gctcaacggc 480ctcaacctac tactgggctg cttcctaatg caggagtcgc
ataagggaga gcgtcgaccg 540atgcccttga gagccttcaa cccagtcagc
tccttccggt gggcgcgggg catgactatc 600gtcgccgcac ttatgactgt
cttctttatc atgcaactcg taggacaggt gccggcagcg 660ctctgggtca
ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
720cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
tcccgccacc 780aaacgtttcg gcgagaagca ggccattatc gccggcatgg
cggccgacgc gctgggctac 840gtcttgctgg cgttcgcgac gcgaggctgg
atggccttcc ccattatgat tcttctcgct 900tccggcggca tcgggatgcc
cgcgttgcag gccatgctgt ccaggcaggt agatgacgac 960catcagggac
agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga
1020ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg
gttggcatgg 1080attgtaggcg ccgccctata ccttgtctgc ctccccgcgt
tgcgtcgcgg tgcatggagc 1140cgggccacct cgacctgaat ggaagccggc
ggcacctcgc taacggattc accactccaa 1200gaattggagc caatcaattc
ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga 1260acatatccat
cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg
1320ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
ctaggctggc 1380ggggttgcct tactggttag cagaatgaat caccgatacg
cgagcgaacg tgaagcgact 1440gctgctgcaa aacgtctgcg acctgagcaa
caacatgaat ggtcttcggt ttccgtgttt 1500cgtaaagtct ggaaacgcgg
aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 1560caggatgctg
ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
1620gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
ttaccctcac 1680aacgttccag taaccgggca tgttcatcat cagtaacccg
tatcgtgagc atcctctctc 1740gtttcatcgg tatcattacc cccatgaaca
gaaattcccc cttacacgga ggcatcaagt 1800gaccaaacag gaaaaaaccg
cccttaacat ggcccgcttt atcagaagcc agacattaac 1860gcttctggag
aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
1920tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
atgacggtga 1980aaacctctga cacatgcagc tcccggagac ggtcacagct
tgtctgtaag cggatgccgg 2040gagcagacaa gcccgtcagg gcgcgtcagc
gggtgttggc gggtgtcggg gcgcagccat 2100gacccagtca cgtagcgata
gcggagtgta tactggctta
actatgcggc atcagagcag 2160attgtactga gagtgcacca tatgcggtgt
gaaataccgc acagatgcgt aaggagaaaa 2220taccgcatca ggcgctcttc
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 2280ctgcggcgag
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
2340gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
ccgtaaaaag 2400gccgcgttgc tggcgttttt ccataggctc cgcccccctg
acgagcatca caaaaatcga 2460cgctcaagtc agaggtggcg aaacccgaca
ggactataaa gataccaggc gtttccccct 2520ggaagctccc tcgtgcgctc
tcctgttccg accctgccgc ttaccggata cctgtccgcc 2580tttctccctt
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
2640gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
gcccgaccgc 2700tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
taagacacga cttatcgcca 2760ctggcagcag ccactggtaa caggattagc
agagcgaggt atgtaggcgg tgctacagag 2820ttcttgaagt ggtggcctaa
ctacggctac actagaagga cagtatttgg tatctgcgct 2880ctgctgaagc
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
2940accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga 3000tctcaagaag atcctttgat cttttctacg gggtctgacg
ctcagtggaa cgaaaactca 3060cgttaaggga ttttggtcat gagattatca
aaaaggatct tcacctagat ccttttaaat 3120taaaaatgaa gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac 3180caatgcttaa
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
3240gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
tggccccagt 3300gctgcaatga taccgcgaga cccacgctca ccggctccag
atttatcagc aataaaccag 3360ccagccggaa gggccgagcg cagaagtggt
cctgcaactt tatccgcctc catccagtct 3420attaattgtt gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3480gttgccattg
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
3540tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
aaaagcggtt 3600agctccttcg gtcctccgat cgttgtcaga agtaagttgg
ccgcagtgtt atcactcatg 3660gttatggcag cactgcataa ttctcttact
gtcatgccat ccgtaagatg cttttctgtg 3720actggtgagt actcaaccaa
gtcattctga gaatagtgta tgcggcgacc gagttgctct 3780tgcccggcgt
caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
3840attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
gagatccagt 3900tcgatgtaac ccactcgtgc acccaactga tcttcagcat
cttttacttt caccagcgtt 3960tctgggtgag caaaaacagg aaggcaaaat
gccgcaaaaa agggaataag ggcgacacgg 4020aaatgttgaa tactcatact
cttccttttt caatattatt gaagcattta tcagggttat 4080tgtctcatga
gcggatacat atttgaatgt atttagaaaa ataaacaaaa gagtttgtag
4140aaacgcaaaa aggccatccg tcaggatggc cttctgctta atttgatgcc
tggcagttta 4200tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
gcaacgttca aatccgctcc 4260cggcggattt gtcctactca ggagagcgtt
caccgacaaa caacagataa aacgaaaggc 4320ccagtctttc gactgagcct
ttcgttttat ttgatgcctg gcagttccct actctcgcat 4380ggggagaccc
cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt
4440caggtgggac caccgcgcta ctgccgccag gcaaattctg ttttatcaga
ccgcttctgc 4500gttctgattt aatctgtatc aggctgaaaa tcttctctca
tccgccaaaa cagaagcttg 4560gctgcaggtc gacggatccc cgggaa
458628425DNAartificial sequenceTerminator Sequence 28ctgttttggc
ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag 60cggtctgata
aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
120gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
cccatgcgag 180agtagggaac tgccaggcat caaataaaac gaaaggctca
gtcgaaagac tgggcctttc 240gttttatctg ttgtttgtcg gtgaacgctc
tcctgagtag gacaaatccg ccgggagcgg 300atttgaacgt tgcgaagcaa
cggcccggag ggtggcgggc aggacgcccg ccataaactg 360ccaggcatca
aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa 420ctctt
42529816DNAartificial sequenceResistance gene 29ttagaaaaac
tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 60accatatttt
tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
120taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
caatacaacc 180tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
aaatcaccat gagtgacgac 240tgaatccggt gagaatggca aaagcttatg
catttctttc cagacttgtt caacaggcca 300gccattacgc tcgtcatcaa
aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 360cgcctgagcg
agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
420atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
aatcaggata 480ttcttctaat acctggaatg ctgttttccc ggggatcgca
gtggtgagta accatgcatc 540atcaggagta cggataaaat gcttgatggt
cggaagaggc ataaattccg tcagccagtt 600tagtctgacc atctcatctg
taacatcatt ggcaacgcta cctttgccat gtttcagaaa 660caactctggc
gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
720attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
ttaatcgcgg 780cctcgagcaa gacgtttccc gttgaatatg gctcat
816304963DNAartificial sequencePlasmid 30ggctgtgcag gtcgtaaatc
actgcataat tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga
catcataacg gttctggcaa atattctgaa atgagctgtt 120gacaattaat
catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
180ggaaacagaa ttctaaggag gaaaaaaaaa tggcaaataa gccaatgcaa
ccgatcacat 240ctacagcaaa taaaattgtg tggtcggatc caactcgttt
atcaactaca ttttcagcaa 300gtctgttacg ccaacgtgtt aaagttggta
tagccgaact gaataatgtt tcaggtcaat 360atgtatctgt ttataagcgt
cctgcaccta aaccggaagg ttgtgcagat gcctgtgtca 420ttatgccgaa
tgaaaaccaa tccattcgca cagtgatttc agggtcagcc gaaaacttgg
480ctaccttaaa agcagaatgg gaaactcaca aacgtaacgt tgacacactc
ttcgcgagcg 540gcaacgccgg tttgggtttc cttgacccta ctgcggctat
cgtatcgtct gatactactg 600cttaatgaag cttggctgtt ttggcggatg
agagaagatt ttcagcctga tacagattaa 660atcagaacgc agaagcggtc
tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 720cccacctgac
cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
780gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
gctcagtcga 840aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
cgctctcctg agtaggacaa 900atccgccggg agcggatttg aacgttgcga
agcaacggcc cggagggtgg cgggcaggac 960gcccgccata aactgccagg
catcaaatta agcagaaggc catcctgacg gatggccttt 1020ttgcgtttct
acaaactctt ttgtttattt ttctagagcc acgttgtgtc tcaaaatctc
1080tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
gtctgcttac 1140ataaacagta atacaaggag tgttatgagc catattcaac
gggaaacgtc ttgctcgagg 1200ccgcgattaa attccaacat ggatgctgat
ttatatgggt ataaatgggc tcgcgataat 1260gtcgggcaat caggtgcgac
aatctatcga ttgtatggga agcccgatgc gccagagttg 1320tttctgaaac
atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
1380aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
tactcctgat 1440gatgcatggt tactcaccac tgcgatcccc gggaaaacag
cattccaggt attagaagaa 1500tatcctgatt caggtgaaaa tattgttgat
gcgctggcag tgttcctgcg ccggttgcat 1560tcgattcctg tttgtaattg
tccttttaac agcgatcgcg tatttcgtct cgctcaggcg 1620caatcacgaa
tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
1680tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc
accggattca 1740gtcgtcactc atggtgattt ctcacttgat aaccttattt
ttgacgaggg gaaattaata 1800ggttgtattg atgttggacg agtcggaatc
gcagaccgat accaggatct tgccatccta 1860tggaactgcc tcggtgagtt
ttctccttca ttacagaaac ggctttttca aaaatatggt 1920attgataatc
ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
1980acgcgtgacc aagtttactc atatgtactt tagattgatt taaaacttca
tttttaattt 2040aaaaggatct aggtgaagat cctttttgat aatctcatga
ccaaaatccc ttaacgtgag 2100ttttcgttcc actgagcgtc agaccccgta
gaaaagatca aaggatcttc ttgagatcct 2160ttttttctgc gcgtaatctg
ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 2220tgtttgccgg
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
2280cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
caagaactct 2340gtagcaccgc ctacatacct cgctctgcta atcctgttac
cagtggctgc tgccagtggc 2400gataagtcgt gtcttaccgg gttggactca
agacgatagt taccggataa ggcgcagcgg 2460tcgggctgaa cggggggttc
gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 2520ctgagatacc
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
2580gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
gctcccaggg 2640ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
acctctgact tgagcgtcga 2700tttttgtgat gctcgtcagg ggggcggagc
ctatggaaaa acgccagcaa cgcggccttt 2760ttacggttcc tggccttttg
ctggcctttt gctcacatgt tctttcctgc gttatcccct 2820gattctgtgg
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
2880acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
gcggtatttt 2940ctccttacgc atctgtgcgg tatttcacac cgcatatggt
gcactctcag tacaatctgc 3000tctgatgccg catagttaag ccagtataca
ctccgctatc gctacgtgac tgggtcatgg 3060ctgcgccccg acacccgcca
acacccgctg acgcgccctg acgggcttgt ctgctcccgg 3120catccgctta
cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
3180cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
tcgtgaagcg 3240attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
gagtttctcc agaagcgtta 3300atgtctggct tctgataaag cgggccatgt
taagggcggt tttttcctgt ttggtcactg 3360atgcctccgt gtaaggggga
tttctgttca tgggggtaat gataccgatg aaacgagaga 3420ggatgctcac
gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
3480gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
ggtcaatgcc 3540agcgcttcgt taatacagat gtaggtgttc cacagggtag
ccagcagcat cctgcgatgc 3600agatccggaa cataatggtg cagggcgctg
acttccgcgt ttccagactt tacgaaacac 3660ggaaaccgaa gaccattcat
gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc 3720ttcacgttcg
ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
3780ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
gacccaacgc 3840tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
cgcgatggat atgttctgcc 3900aagggttggt ttgcgcattc acagttctcc
gcaagaattg attggctcca attcttggag 3960tggtgaatcc gttagcgagg
tgccgccggc ttccattcag gtcgaggtgg cccggctcca 4020tgcaccgcga
cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc
4080caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca
gcggtccaat 4140gatcgaagtt aggctggtaa gagccgcgag cgatccttga
agctgtccct gatggtcgtc 4200atctacctgc ctggacagca tggcctgcaa
cgcgggcatc ccgatgccgc cggaagcgag 4260aagaatcata atggggaagg
ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc 4320cagcgcgtcg
gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
4380gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
caagcgacag 4440gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
aaaatgaccc agagcgctgc 4500cggcacctgt cctacgagtt gcatgataaa
gaagacagtc ataagtgcgg cgacgatagt 4560catgccccgc gcccaccgga
aggagctgac tgggttgaag gctctcaagg gcatcggtcg 4620acgctctccc
ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
4680gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
gtcccccggc 4740cacggggcct gccaccatac ccacgccgaa acaagcgctc
atgagcccga agtggcgagc 4800ccgatcttcc ccatcggtga tgtcggcgat
ataggcgcca gcaaccgcac ctgtggcgcc 4860ggtgatgccg gccacgatgc
gtccggcgta gaggatccgg gcttatcgac tgcacggtgc 4920accaatgctt
ctggcgtcag gcagccatcg gaagctgtgg tat 4963316DNAartificial
sequenceStop Codon 31tgaaca 6326DNAartificial sequenceStop Codon
32taatga 6334525DNAartificial sequencePlasmid 33tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt cagacatgca tttcatcctt
420agttactaag cacgaggaac gactatcacg gcttgaatag gacatttagt
cttatcaaac 480ttagtgaagt caaacggtat ggcaccacca ctggatggat
cgcgccaaaa gccaactatc 540acgccatcgg cgtgataggc cgcaatatcg
ctaagagagc aataagcatt tatcgactta 600agataaatga atcgctcaat
gttaccgaaa gcaccaggtt tcttgccctc gcgaatatca 660tacttctgat
cacgcatagc ctgatcagta gcaagataag tcgcatcaag gtcaatataa
720ccattgccac ggcaaccgcg gaacgtggta taactaagcc gagaatccca
atcacgccac 780tttgtattgc ccaaaagatc tttgagggca acatcaaatt
cgcgaggctg gagttcaaca 840gcattataga taggccacgg tcggttctta
gtaggaggct cgtaaacctc ctctagggac 900caaattgcga agggacaggt
atacttacct gtccctggcg gcggatcaat cggtggatcc 960ggaataaccg
gatcgggttt tgaccctgag ccaccaccgg caatgagcag tcattaatac
1020gctgggttca gctgatcaat agcatcgatc agcagaggac tagcgagcag
agcagcaagc 1080tctgtacgaa caaaagctcg ttcctcatcg gtactatact
gcgtgaacga aaaggtcacg 1140tcagcatatg cctggcgagt aacggatggg
tcacaagaac cgtttgcagt gcaagcggtc 1200gggttctgga tcttaacctg
gaccttgtag ttcttacgat tgcgagaagg ctgagatacc 1260gaaacggtaa
cacgcttctc cagcgcagga actgcacccg cttgtgaaag cgaggcaacg
1320ccgttagtgg gatttacccc acgcggattg aggaccagag tttgttttcc
atctttcccg 1380atgttaccta aagtaacagt ctctaatttt gccatcgttt
tttacctcct tctagagtca 1440ttatggtttt gccatacatc agtatggtgt
agcagcactt attataatct ttattgcctc 1500ttaaaactta atccacatca
aaactcaaat acttttaacc ccagcgtcct gtaagctctg 1560cattaatgaa
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
1620tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
atcagctcac 1680tcaaaggcgg taatacggtt atccacagaa tcaggggata
acgcaggaaa gaacatgtga 1740gcaaaaggcc agcaaaaggc caggaaccgt
aaaaaggccg cgttgctggc gtttttccat 1800aggctccgcc cccctgacga
gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 1860ccgacaggac
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
1920gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
aagcgtggcg 1980ctttctcata gctcacgctg taggtatctc agttcggtgt
aggtcgttcg ctccaagctg 2040ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg taactatcgt 2100cttgagtcca acccggtaag
acacgactta tcgccactgg cagcagccac tggtaacagg 2160attagcagag
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
2220ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
taccttcgga 2280aaaagagttg gtagctcttg atccggcaaa caaaccaccg
ctggtagcgg tggttttttt 2340gtttgcaagc agcagattac gcgcagaaaa
aaaggatctc aagaagatcc tttgatcttt 2400tctacggggt ctgacgctca
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 2460ttatcaaaaa
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
2520taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
tgaggcacct 2580atctcagcga tctgtctatt tcgttcatcc atagttgcct
gactccccgt cgtgtagata 2640actacgatac gggagggctt accatctggc
cccagtgctg caatgatacc gcgagaccca 2700cgctcaccgg ctccagattt
atcagcaata aaccagccag ccggaagggc cgagcgcaga 2760agtggtcctg
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
2820gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
aggcatcgtg 2880gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
gttcccaacg atcaaggcga 2940gttacatgat cccccatgtt gtgcaaaaaa
gcggttagct ccttcggtcc tccgatcgtt 3000gtcagaagta agttggccgc
agtgttatca ctcatggtta tggcagcact gcataattct 3060cttactgtca
tgccatccgt aagatgcttt tctgtgactg gtgagtgggg ggggggggcg
3120ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa
tcgccccatc 3180atccagccag aaagtgaggg agccacggtt gatgagagct
ttgttgtagg tggaccagtt 3240ggtgattttg aacttttgct ttgccacgga
acggtctgcg ttgtcgggaa gatgcgtgat 3300ctgatccttc aactcagcaa
aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc 3360agcgtaatgc
tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg
3420agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
tttttgaaaa 3480agccgtttct gtaatgaagg agaaaactca ccgaggcagt
tccataggat ggcaagatcc 3540tggtatcggt ctgcgattcc gactcgtcca
acatcaatac aacctattaa tttcccctcg 3600tcaaaaataa ggttatcaag
tgagaaatca ccatgagtga cgactgaatc cggtgagaat 3660ggcaaaagct
tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
3720tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
agcgagacga 3780aatacgcgat cgctgttaaa aggacaatta caaacaggaa
tcgaatgcaa ccggcgcagg 3840aacactgcca gcgcatcaac aatattttca
cctgaatcag gatattcttc taatacctgg 3900aatgctgttt tcccggggat
cgcagtggtg agtaaccatg catcatcagg agtacggata 3960aaatgcttga
tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
4020tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
tggcgcatcg 4080ggcttcccat acaatcgata gattgtcgca cctgattgcc
cgacattatc gcgagcccat 4140ttatacccat ataaatcagc atccatgttg
gaatttaatc gcggcctcga gcaagacgtt 4200tcccgttgaa tatggctcat
aacacccctt gtattactgt ttatgtaagc agacagtttt 4260attgttcatg
atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca
4320acgtggcttt cccccccccc ccattattga agcatttatc agggttattg
tctcatgagc 4380ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
gggttccgcg cacatttccc 4440cgaaaagtgc cacctgacgt ctaagaaacc
attattatca tgacattaac ctataaaaat 4500aggcgtatca cgaggccctt tcgtc
45253456DNAartificial sequencePrimer Sequence 34gcgcgcgaat
tcaggaggta aaaaacgatg gcaaaattag agactgttac tttagg
563533DNAartificial sequencePrimer Sequence 35gcatgcaagc ttagacatgc
atttcatcct tag 333657DNAartificial sequencePrimer Sequence
36gcgcgcgaat tctaaggagg aaaaaaaaat ggcaaaatta gagactgtta ctttagg
57373914DNAartificial sequenceCloning Vector 37tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt ccccggatcc gtcgacctgc
420aggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact
cataccaggc 480ctgaatcgcc ccatcatcca gccagaaagt gagggagcca
cggttgatga gagctttgtt 540gtaggtggac cagttggtga ttttgaactt
ttgctttgcc acggaacggt ctgcgttgtc 600gggaagatgc gtgatctgat
ccttcaactc agcaaaagtt cgatttattc aacaaagccg 660ccgtcccgtc
aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat
720tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
attatcaata 780ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
actcaccgag gcagttccat 840aggatggcaa gatcctggta tcggtctgcg
attccgactc gtccaacatc aatacaacct 900attaatttcc cctcgtcaaa
aataaggtta tcaagtgaga aatcaccatg agtgacgact 960gaatccggtg
agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
1020ccattacgct cgtcatcaaa
atcactcgca tcaaccaaac cgttattcat tcgtgattgc 1080gcctgagcga
gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
1140tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
atcaggatat 1200tcttctaata cctggaatgc tgttttcccg gggatcgcag
tggtgagtaa ccatgcatca 1260tcaggagtac ggataaaatg cttgatggtc
ggaagaggca taaattccgt cagccagttt 1320agtctgacca tctcatctgt
aacatcattg gcaacgctac ctttgccatg tttcagaaac 1380aactctggcg
catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
1440ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
taatcgcggc 1500ctcgagcaag acgtttcccg ttgaatatgg ctcataacac
cccttgtatt actgtttatg 1560taagcagaca gttttattgt tcatgatgat
atatttttat cttgtgcaat gtaacatcag 1620agattttgag acacaacgtg
gctttccccc ccccccctgc aggtcgacgg atccggggaa 1680ttcgtaatca
tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
1740caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
tgagctaact 1800cacattaatt gcgttgcgct cactgcccgc tttccagtcg
ggaaacctgt cgtgccagct 1860gcattaatga atcggccaac gcgcggggag
aggcggtttg cgtattgggc gctcttccgc 1920ttcctcgctc actgactcgc
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 1980ctcaaaggcg
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
2040agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
cgtttttcca 2100taggctccgc ccccctgacg agcatcacaa aaatcgacgc
tcaagtcaga ggtggcgaaa 2160cccgacagga ctataaagat accaggcgtt
tccccctgga agctccctcg tgcgctctcc 2220tgttccgacc ctgccgctta
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2280gctttctcaa
tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
2340gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
gtaactatcg 2400tcttgagtcc aacccggtaa gacacgactt atcgccactg
gcagcagcca ctggtaacag 2460gattagcaga gcgaggtatg taggcggtgc
tacagagttc ttgaagtggt ggcctaacta 2520cggctacact agaaggacag
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2580aaaaagagtt
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
2640tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
ctttgatctt 2700ttctacgggg tctgacgctc agtggaacga aaactcacgt
taagggattt tggtcatgag 2760attatcaaaa aggatcttca cctagatcct
tttaaattaa aaatgaagtt ttaaatcaat 2820ctaaagtata tatgagtaaa
cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2880tatctcagcg
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
2940aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
cgcgagaccc 3000acgctcaccg gctccagatt tatcagcaat aaaccagcca
gccggaaggg ccgagcgcag 3060aagtggtcct gcaactttat ccgcctccat
ccagtctatt aattgttgcc gggaagctag 3120agtaagtagt tcgccagtta
atagtttgcg caacgttgtt gccattgcta caggcatcgt 3180ggtgtcacgc
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
3240agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
ctccgatcgt 3300tgtcagaagt aagttggccg cagtgttatc actcatggtt
atggcagcac tgcataattc 3360tcttactgtc atgccatccg taagatgctt
ttctgtgact ggtgagtact caaccaagtc 3420attctgagaa tagtgtatgc
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3480taccgcgcca
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
3540aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
ctcgtgcacc 3600caactgatct tcagcatctt ttactttcac cagcgtttct
gggtgagcaa aaacaggaag 3660gcaaaatgcc gcaaaaaagg gaataagggc
gacacggaaa tgttgaatac tcatactctt 3720cctttttcaa tattattgaa
gcatttatca gggttattgt ctcatgagcg gatacatatt 3780tgaatgtatt
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
3840acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
ggcgtatcac 3900gaggcccttt cgtc 39143857DNAartificial sequencePrimer
sequence 38gcgcgcgaat tctaaggagg aaaaaaaaat ggcaaataag ccaatgcaac
cgatcac 573944DNAartificial sequencePrimer Sequence 39gcatgcaagc
ttcattaagc agtagtatca gacgatacga tagc 44404962DNAartificial
sequenceExpression Construct 40ggctgtgcag gtcgtaaatc actgcataat
tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga catcataacg
gttctggcaa atattctgaa atgagctgtt 120gacaattaat catcggctcg
tataatgtgt ggaattgtga gcggataaca atttcacaca 180ggaaacagaa
ttcaggaggt aaaaaacgat ggcaaataag ccaatgcaac cgatcacatc
240tacagcaaat aaaattgtgt ggtcggatcc aactcgttta tcaactacat
tttcagcaag 300tctgttacgc caacgtgtta aagttggtat agccgaactg
aataatgttt caggtcaata 360tgtatctgtt tataagcgtc ctgcacctaa
accggaaggt tgtgcagatg cctgtgtcat 420tatgccgaat gaaaaccaat
ccattcgcac agtgatttca gggtcagccg aaaacttggc 480taccttaaaa
gcagaatggg aaactcacaa acgtaacgtt gacacactct tcgcgagcgg
540caacgccggt ttgggtttcc ttgaccctac tgcggctatc gtatcgtctg
atactactgc 600ttaatgaagc ttggctgttt tggcggatga gagaagattt
tcagcctgat acagattaaa 660tcagaacgca gaagcggtct gataaaacag
aatttgcctg gcggcagtag cgcggtggtc 720ccacctgacc ccatgccgaa
ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg 780tctccccatg
cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
840agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
gtaggacaaa 900tccgccggga gcggatttga acgttgcgaa gcaacggccc
ggagggtggc gggcaggacg 960cccgccataa actgccaggc atcaaattaa
gcagaaggcc atcctgacgg atggcctttt 1020tgcgtttcta caaactcttt
tgtttatttt tctagagcca cgttgtgtct caaaatctct 1080gatgttacat
tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
1140taaacagtaa tacaaggagt gttatgagcc atattcaacg ggaaacgtct
tgctcgaggc 1200cgcgattaaa ttccaacatg gatgctgatt tatatgggta
taaatgggct cgcgataatg 1260tcgggcaatc aggtgcgaca atctatcgat
tgtatgggaa gcccgatgcg ccagagttgt 1320ttctgaaaca tggcaaaggt
agcgttgcca atgatgttac agatgagatg gtcagactaa 1380actggctgac
ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
1440atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
ttagaagaat 1500atcctgattc aggtgaaaat attgttgatg cgctggcagt
gttcctgcgc cggttgcatt 1560cgattcctgt ttgtaattgt ccttttaaca
gcgatcgcgt atttcgtctc gctcaggcgc 1620aatcacgaat gaataacggt
ttggttgatg cgagtgattt tgatgacgag cgtaatggct 1680ggcctgttga
acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag
1740tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
aaattaatag 1800gttgtattga tgttggacga gtcggaatcg cagaccgata
ccaggatctt gccatcctat 1860ggaactgcct cggtgagttt tctccttcat
tacagaaacg gctttttcaa aaatatggta 1920ttgataatcc tgatatgaat
aaattgcagt ttcatttgat gctcgatgag tttttctaaa 1980cgcgtgacca
agtttactca tatgtacttt agattgattt aaaacttcat ttttaattta
2040aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
taacgtgagt 2100tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
aggatcttct tgagatcctt 2160tttttctgcg cgtaatctgc tgcttgcaaa
caaaaaaacc accgctacca gcggtggttt 2220gtttgccgga tcaagagcta
ccaactcttt ttccgaaggt aactggcttc agcagagcgc 2280agataccaaa
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
2340tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
gccagtggcg 2400ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
accggataag gcgcagcggt 2460cgggctgaac ggggggttcg tgcacacagc
ccagcttgga gcgaacgacc tacaccgaac 2520tgagatacct acagcgtgag
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 2580acaggtatcc
ggtaagcggc agggtcggaa caggagagcg cacgagggag ctcccagggg
2640gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
gagcgtcgat 2700ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
cgccagcaac gcggcctttt 2760tacggttcct ggccttttgc tggccttttg
ctcacatgtt ctttcctgcg ttatcccctg 2820attctgtgga taaccgtatt
accgcctttg agtgagctga taccgctcgc cgcagccgaa 2880cgaccgagcg
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
2940tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
acaatctgct 3000ctgatgccgc atagttaagc cagtatacac tccgctatcg
ctacgtgact gggtcatggc 3060tgcgccccga cacccgccaa cacccgctga
cgcgccctga cgggcttgtc tgctcccggc 3120atccgcttac agacaagctg
tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 3180gtcatcaccg
aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
3240ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
gaagcgttaa 3300tgtctggctt ctgataaagc gggccatgtt aagggcggtt
ttttcctgtt tggtcactga 3360tgcctccgtg taagggggat ttctgttcat
gggggtaatg ataccgatga aacgagagag 3420gatgctcacg atacgggtta
ctgatgatga acatgcccgg ttactggaac gttgtgaggg 3480taaacaactg
gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
3540gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
ctgcgatgca 3600gatccggaac ataatggtgc agggcgctga cttccgcgtt
tccagacttt acgaaacacg 3660gaaaccgaag accattcatg ttgttgctca
ggtcgcagac gttttgcagc agcagtcgct 3720tcacgttcgc tcgcgtatcg
gtgattcatt ctgctaacca gtaaggcaac cccgccagcc 3780tagccgggtc
ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
3840gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
tgttctgcca 3900agggttggtt tgcgcattca cagttctccg caagaattga
ttggctccaa ttcttggagt 3960ggtgaatccg ttagcgaggt gccgccggct
tccattcagg tcgaggtggc ccggctccat 4020gcaccgcgac gcaacgcggg
gaggcagaca aggtataggg cggcgcctac aatccatgcc 4080aacccgttcc
atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
4140atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
atggtcgtca 4200tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
cgatgccgcc ggaagcgaga 4260agaatcataa tggggaaggc catccagcct
cgcgtcgcga acgccagcaa gacgtagccc 4320agcgcgtcgg ccgccatgcc
ggcgataatg gcctgcttct cgccgaaacg tttggtggcg 4380ggaccagtga
cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
4440ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
gagcgctgcc 4500ggcacctgtc ctacgagttg catgataaag aagacagtca
taagtgcggc gacgatagtc 4560atgccccgcg cccaccggaa ggagctgact
gggttgaagg ctctcaaggg catcggtcga 4620cgctctccct tatgcgactc
ctgcattagg aagcagccca gtagtaggtt gaggccgttg 4680agcaccgccg
ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
4740acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
gtggcgagcc 4800cgatcttccc catcggtgat gtcggcgata taggcgccag
caaccgcacc tgtggcgccg 4860gtgatgccgg ccacgatgcg tccggcgtag
aggatccggg cttatcgact gcacggtgca 4920ccaatgcttc tggcgtcagg
cagccatcgg aagctgtggt at 4962
* * * * *
References