U.S. patent application number 10/215060 was filed with the patent office on 2003-06-19 for methods of preserving prokaryotic cells and compositions obtained thereby.
Invention is credited to Colaco, Camilo, Dhaliwal, Kamaljit S., Roser, Bruce J., Tunnacliffe, Alan G., Welsh, David T..
Application Number | 20030113900 10/215060 |
Document ID | / |
Family ID | 21864890 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113900 |
Kind Code |
A1 |
Tunnacliffe, Alan G. ; et
al. |
June 19, 2003 |
Methods of preserving prokaryotic cells and compositions obtained
thereby
Abstract
This invention provides methods of drying and stabilizing
prokaryotic cells, and the compositions obtained thereby. The cells
are first cultured or incubated under conditions sufficient to
induce intracellular trehalose, suspended in a stabilizing solution
and dried to form a solid glass. The resulting product is
storage-stable at room temperature, showing little viability loss
on storage.
Inventors: |
Tunnacliffe, Alan G.; (
Horningsea, GB) ; Welsh, David T.; (Stanley, GB)
; Roser, Bruce J.; (Cambridge, GB) ; Dhaliwal,
Kamaljit S.; (Hitchin, GB) ; Colaco, Camilo;
(Cambridge, GB) |
Correspondence
Address: |
Madeline I. Johnston
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304
US
|
Family ID: |
21864890 |
Appl. No.: |
10/215060 |
Filed: |
August 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10215060 |
Aug 7, 2002 |
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08985343 |
Dec 4, 1997 |
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6468782 |
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60032423 |
Dec 5, 1996 |
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Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
C12N 1/04 20130101; A61K
35/741 20130101; A61K 39/02 20130101; A61K 35/742 20130101 |
Class at
Publication: |
435/252.1 |
International
Class: |
C12N 001/20 |
Claims
We claim:
1. A method of preserving prokaryotic cells comprising the steps
of: a) increasing intracellular trehalose concentration in the
prokaryotic cells to an amount effective to increase storage
stability; b) mixing the prokaryotic cells obtained in step a) with
a drying solution comprising a stabilizing agent; and c) drying the
product of step b) under conditions sufficient to produce a glass
form of the stabilizing agent having less than about 5% residual
moisture.
2. The method according to claim 1, wherein the method of
increasing intracellular trehalose concentration is selected from
the group consisting of culturing in an osmolarity sufficient to
increase intracellular trehalose production, expressing a
recombinant trehalose synthase gene or genes and introducing
exogenous trehalose.
3. The method according to claim 2, wherein the osmolarity is at
least about 350 mOsmoles -1.5 Osmoles
4. The method according to claim 2, wherein the osmolarity is at
least about 400 mOsmoles -1 Osmole.
5. The method according to claim 2, wherein the osmolarity is at
least about 300 mOsmoles.
6. The method according to claim 2, wherein the osmolarity is at
least about 500 mOsmoles.
7. The method according to claim 2, wherein the osmolarity is
increased by adding at least one salt wherein the salt is selected
from the group consisting of Na.sub.2PO.sub.4, KH.sub.2PO.sub.4,
NH.sub.4Cl, NaCl, MgSO.sub.4, CaCl.sub.2, thiamine HCl, or any
combination thereof.
8. The method according to claim 1, wherein the prokaryotic cells
are bacteria.
9. The method according to claim 8, wherein the bacteria are
selected from the group consisting of Escherichia, Bacillus,
Salmonella, or Vibrio.
10. The method according to claim 1, wherein the stabilizing agent
is trehalose.
11. The method according to claim 10, wherein the intracellular
concentration of trehalose is at least about 100 mM.
12. The method according to claim 1, wherein the stabilizing agent
is a non-reducing carbohydrate.
13. The method according to claim 12, wherein the drying solution
comprises at least about 25% non-reducing carbohydrate.
14. The method according to claim 12, wherein the drying solution
comprises at least about 45% non-reducing carbohydrate.
15. The method according to claim 12, wherein the non-reducing
carbohydrate is selected from the group consisting of trehalose,
maltitol (4-O-.beta.-D-glucopyranosyl-D-glucitol), lactitol
(4-O-.beta.-D-galactopyranosyl-D-glucitol), palatinit [a mixture of
GPS (.alpha.-D-glucopyranosyl-1.fwdarw.6-sorbitol) and GPM
(.alpha.-D-glucopyranosyl-1.fwdarw.6-mannitol)], GPS, GPM and
hydrogenated maltooligosaccharides and maltooligosaccharides.
16. The method according to claim 1, wherein the drying comprises
the following steps: a) evaporating the solution to obtain a syrup;
b) exposing the syrup to a pressure reduced external pressure and
temperature sufficient to cause boiling of the syrup; and c)
removing moisture so that residual moisture does not exceed about
5%.
17. The method according to claim 16, wherein the vacuum is
initially about 30 mT with an initial temperature of about
40.degree. C.
18. The method according to claim 1, wherein step c) further
comprises the steps of i) holding the temperature at about
40.degree. C. for about 16 hours; and ii) raising the temperature
incrementally to about 80.degree. C. at a rate of about 2.5.degree.
C. per minute at increment of about 2.degree. C., wherein each
increment is of a duration of about 12 minutes.
19. The method according to claim 1, where the glass has a residual
moisture that does not exceed about 2.5%.
20. The method according to claim 1 wherein the glass is a foamed
glass matrix.
21. A composition obtained according to the method of claim 1.
22. The method according to claim 21, wherein the solvent is
aqueous.
23. The method according to claim 1, further comprising the step
of: d) reconstituting the prokaryotic cells by adding a suitable
solvent.
24. A method for reconstituting dried, stabilized prokaryotic cells
comprising adding a suitable solvent to the dried prokaryotic cells
obtained in claim 1 in an amount sufficient to attain viability.
Description
RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Serial No. 60/032,423 and its filing date
of Dec. 5, 1996.
TECHNICAL FIELD
[0002] This invention relates to the field of preserving cells.
More specifically, it relates to methods of drying and stabilizing
prokaryotic cells and the compositions obtained thereby.
BACKGROUND ART
[0003] Live prokaryotic cells, particularly bacteria, are widely
and increasingly used in important medical, agricultural and
industrial applications. Agricultural, or environmental,
applications include biopesticides and bioremediation. Medical
applications include use of live bacteria in vaccines as well as
production of pharmaceutical products and numerous industrial
compositions. The use of live bacterial vaccines promises only to
increase, given the dramatic rise in biotechnology as well as the
intensive research into the treatment of infectious diseases over
the past twenty years.
[0004] Bacterial cells must be able to be stored for significant
periods of time while preserving their viability to be used
effectively both in terms of desired results and cost. Storage
viability has proven to be a major difficulty. Methods for
preserving live prokaryotic cells suffer from several serious
drawbacks, such as being energy-intensive and requiring cold
storage. Furthermore, existing preservation methods fail to provide
satisfactory viability upon storage, especially if cells are stored
at ambient or higher temperature.
[0005] Freeze-drying is often used for preservation and storage of
prokaryotic cells. However, it has the undesirable characteristics
of significantly reducing viability as well as being time- and
energy-intensive and thus expensive. Freeze-drying involves placing
the cells in solution, freezing the solution, and exposing the
frozen solid to a vacuum under conditions where it remains solid
and the water and any other volatile components are removed by
sublimation. The resulting dried formulation comprises the
prokaryotic cells.
[0006] In spite of the apparent ubiquity of freeze-drying,
freeze-dried bacteria are unstable at ambient temperatures, thus
necessitating storage by refrigeration. Even when refrigerated,
however, the cells can quickly lose viability. Damage caused by
this process may be circumvented, to a certain degree, by the use
of excipients such as lyoprotectants. However, lyoprotectants may
subsequently react with the dried cells, imposing inherent
instability upon storage of the freeze-dried prokaryotic cells.
[0007] Other methods used to prepare dry, purportedly stable
preparations of prokaryotic cells such as ambient temperature
drying, spray drying, liquid formulations, and freezing of
bacterial cultures with cryoprotectants also have drawbacks. For a
general review on desiccation tolerance of prokaryotes, see Potts
(1994) Micro. Rev. 58:755-805. Ambient temperature drying
techniques eliminate the freezing step and associated freeze-damage
to the substance, and these techniques are more rapid and
energy-efficient in the removal of water. Crowe et al. (1990)
Cryobiol. 27:219-231. However, ambient temperature drying often
yields unsatisfactory viability. Spray drying results in limited
storage time and reduced viability, even when stabilizing
excipients are used. For a general review, see Lievense and van't
Reit (1994) Adv. Biochem. Eng. Biotechnol. 51:45-63; 72-89. Liquid
formulations may provide only short-term stabilization and require
refrigeration. Freezing bacterial cultures results in substantial
damage to the bacterial cell wall and loss of viability which is
only reduced but not eliminated by the use of cryoprotectants.
Moreover, these frozen cultures also need to be stored
refrigerated.
[0008] Trehalose,
(.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside), is a
naturally occurring, non-reducing disaccharide which was initially
found to be associated with the prevention of desiccation damage in
certain plants and animals which can dry out without damage and can
revive when rehydrated. Trehalose has been shown to be useful in
preventing denaturation of proteins, viruses and foodstuffs during
desiccation. See U.S. Pat. Nos. 4,891,319; 5,149,653; 5,026,566;
Blakeley et al. (1990) Lancet 336:854-855; Roser (July 1991) Trends
in Food Sci. and Tech. 10:166-169; Colaco et al. (1992) Biotechnol.
Internat. 1:345-350; Roser (1991) Bio. Pharm. 4:47-53; Colaco et
al. (1992) Bio/Tech. 10:1007-1011; and Roser et al. (May 1993) New
Scientist, pp. 25-28. Trehalose dihydrate is available commercially
in good manufacturing process (GMP) grade crystalline formulations.
A method of making trehalose from starch is described in EP patent
publication No. 639 645 A1. This method involves a two step
enzymatic bioconversion of starch to yield a trehalose syrup from
which the sugar is recovered by crystallisation.
[0009] Bacteria are able to counteract osmotic shock by
accumulating and/or synthesizing potassium with a few types of
organic molecules, including some sugars. Osmoregulation in
bacteria such as Escherichia coli in glucose-mineral medium without
any osmoprotective compounds involves the endogenous production of
trehalose. Larsen et al. (1987) Arch. Microbiol. 147:1-7; Dinnbier
et al. (1988) Arch. Microbiol. 150:348-357; Giaever et al. (1988)
J. Bacteriol. 170:2841-2849; and Welsh et al. (1991) J. Gen.
Microbiol. 137:745-750.
[0010] One method of preserving prokaryotic cells is freeze-drying
in the presence of trehalose. See, e.g., Israeli et al. (1993)
Cryobiol. 30:519-523. However, this method provides unsatisfactory
viability. Israeli et al. freeze dried E. coli in the presence of
100 mM trehalose but reported survival data for only four days
after exposure of the dried samples to air at 21.degree. C. A later
study tested survival rates of E. coli and Bacillus fluoringiensis
freeze-dried in the presence of trehalose. Leslie et al. (1995)
Appl. Env. Microbiol. 61:3592-3597. Survival data were reported
only for 4 days after exposure of the dried samples to air.
[0011] Another study comparing freeze-dried to air-dried (sealed
under nitrogen) E. coli in the presence of trehalose reported
survival rates of about 10.sup.7 to over 10.sup.10 colony forming
units (CFU) per ml for cells stored for 25 weeks, but the cells
were stored at 4.degree. C. Louis et al. (1994) Appl. Microbiol.
Biotechnol. 41:684-688.
[0012] In view of increasing applications for viable bacteria and
the existing problems regarding maintaining bacterial viability
during storage, there is a pressing need for a method to
inexpensively dry and stabilize prokaryotic cells. It is especially
desirable to develop methods that would allow storage of dried
prokaryotic cells at ambient temperature, i.e., not requiring
refrigeration. The methods described herein address this need by
providing dry, remarkably storage-stable, prokaryotic cells that
retain viability without the need for refrigeration.
[0013] All references cited herein are hereby incorporated herein
by reference in their entirety.
SUMMARY OF THE INVENTION
[0014] The present invention encompasses methods of producing
dried, stabilized prokaryotic cells. The invention also includes
compositions produced by these methods, as well as methods of
reconstituting the prokaryotic cells.
[0015] Accordingly, in one aspect, the invention provides methods
of preserving prokaryotic cells, comprising culturing the
prokaryotic cells under conditions which increase intracellular
trehalose concentration to an amount effective to increase storage
stability, mixing the prokaryotic cells with a drying solution
which comprises a stabilizing agent such as trehalose, and drying
the prokaryotic cells such that a glass is produced having less
than about 5% residual moisture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the alignment of trehalose synthase amino acid
sequences encoded by genes from a variety of organisms: 1.
Kluyveromyces lactis; 2. Saccharomyces cerevisiae, 3. Aspergillus
niger; 4. Schizosaccharomyces pombe; 5. Mycobacterium leprae and 6
E. coli (SEQ ID NOS: 1-6, respectively).
[0017] FIG. 2 is a half-tone reproduction of a Southern blot
testing for the presence of trehalose synthase genes in E. coli and
Salmonella. The horizontal lines on the left represent molecular
weight markers (.lambda. Hind III) of 23, 9.3, 6.6, 4.4, 2.3, 2.0
and 0.56 kb, respectively.
[0018] FIG. 3 is a graph depicting stability of E. coli NCIMB 9484
after storage at 37.degree. C. The circles indicate intracellular
trehalose induction and the triangles represent no trehalose
induction.
[0019] FIG. 4 is a graph depicting the relationship between T.sub.g
and residual moisture in a formulation of 45% trehalose and 1.5%
Kollidon 90.
[0020] FIG. 5 is a graph depicting the relationship between
residual moisture and length of drying time in a formulation of 45%
trehalose and 0.1% CMC. The FTS drying protocol was 30 mT ST
40.degree. C. (x hrs).
[0021] FIGS. 6A and 6B are graphs depicting the effect of a high
osmolarity condition (0.5M NaCl) on intracellular trehalose
concentration. FIG. 6A shows the accumulation of intracellular
trehalose concentration and growth curve for E. coli grown at
37.degree. C. in Evans medium and 0.5 M NaCl. FIG. 6B shows
accumulation of intracellular trehalose concentration and growth
curve at 37.degree. C. for E. coli grown at 37.degree. C. in Evans
medium lacking NaCl. In both A and B, the circles represent induced
trehalose concentration and the squares represent cell growth
(absorbance) measured at 600 nm.
[0022] FIG. 7 is a reproduction of a series of tracings from a HPLC
analysis of intracellular trehalose concentration in Salmonella
before and after trehalose induction by osmotic shock.
[0023] FIG. 8 is a graph depicting the relationship between cell
viability and length of drying time in a formulation of 45%
trehalose and 0.1% CMC. The FTS drying protocol was 30 mT ST
40.degree. C. (x hrs).
[0024] FIG. 9 is a graph depicting intracellular trehalose
(.circle-solid.) and protein (.box-solid.) concentration during
growth of S. typhimurium at 37.degree. C.
[0025] FIG. 10 is a graph depicting the percent recovery of
trehalose induced (.circle-solid.) and non-induced
(.tangle-solidup.) S. typhimurium 1344 after storage at 37.degree.
C.
MODES FOR CARRYING OUT THE INVENTION
[0026] We have found that prokaryotic cells can be dried and
stabilized by inducing intracellular trehalose production to an
amount effective to increase storage stability and drying the cells
in the presence of a stabilizing agent. The methods for stabilizing
prokaryotic cells described herein can be used for producing dried,
stable bacteria useful for pharmacological treatment, prophylaxis,
agricultural and industrial applications.
[0027] Prokaryotic cells obtained by the methods disclosed herein
are remarkably stable: bacteria stabilized by these methods retain
high viability even after storage at ambient or above ambient
temperatures. Bacteria dried under these conditions retain about
50-80% viability upon drying. Furthermore, bacteria stabilized by
these methods show less than 10% loss of viability on storage even
after being stored at temperatures up to at least 37.degree. C. for
as long as six weeks. This degree of stabilization during drying
and storage is significantly greater than previously reported using
other methods. The stabilized cells can be stored at room
temperature and thus do not require refrigeration. Depending on the
conditions, drying can generally be accomplished within 24 hours
which provides energy and cost savings as well as increased
viability.
[0028] The methods and compositions of the invention facilitate the
development of many needed, useful products, including, but not
limited to: (i) live bacterial vaccines in a dry stable form; (ii)
live bacterial neutraceuticals in a dry stable form; (iii) other
live bacterial pharmaceutical products in a dry stable form, e.g.,
for treatment of vaginal or urinary tract infections; (iv) live
bacterial starter cultures in a dry stable form for commercial
products such as for the dairy industry; (v) live bacteria in a dry
stable form for agricultural, ecological or bioremedial use, such
as pesticides; and (vi) live bacterial cultures in a dry stable
form for the biotechnology industry.
[0029] As used herein, "prokaryotic cells" are cells that exhibit
characteristics of prokaryotes, which is a term well known in the
art. Prokaryotes are typically unicellular organisms and lack
organelles (such as mitochondria, chloroplasts, and Golgi
apparatus), a cytoskeleton, and a discrete nucleus. Examples of
prokaryotic cells include bacteria, such as eubacteria,
cyanobacteria and prochlorophytes; archaebacteria; and other
microorganisms such as rickettsias, mycoplasmas, spiroplasmas, and
chlamydiae. For purposes of this invention, prokaryotes are capable
of synthesizing trehalose. This ability can be native or conferred
by recombinant techniques. The ability to synthesize trehalose can
be determined by measuring intracellular trehalose concentration,
which is described below. Preferably, the prokaryotic cells are
bacteria. Examples of bacteria include, but are not limited to,
Escherichia, Bacillus(including Lactobacillus), Salmonella, and
Vibrio.
[0030] The stabilizing agents are preferably carbohydrates.
"Carbohydrates" include, but are not limited to, monosaccharides,
disaccharides, trisaccharides, oligosaccharides and their
corresponding sugar alcohols, polyhydroxyl compounds such as
carbohydrate derivatives and chemically modified carbohydrates,
hydroxyethyl starch and sugar copolymers. Both natural and
synthetic carbohydrates are suitable for use herein. Synthetic
carbohydrates include, but are not limited to, those which have the
glycosidic bond replaced by a thiol or carbon bond. Both D and L
forms of the carbohydrates may be used. For purposes of this
invention, the carbohydrate is preferably non-reducing. Preferably,
the non-reducing carbohydrate is trehalose. Other examples of
preferred non-reducing carbohydrates are provided below.
[0031] Conditions that "increase intracellular trehalose
concentration" are conditions that initiate, encourage, allow,
and/or increase the rate of synthesis of trehalose within the
cell(s), and/or increase the amount of trehalose within the cell(s)
when compared to growing or incubating the cell(s) without these
conditions. Conditions (including preferred conditions) that
stimulate production of intracellular production of trehalose are
discussed in detail below. Examples of these conditions include,
but are not limited to, growing the cell(s) under stressful
conditions such as osmotic shock, i.e., high salt conditions.
Conditions that stimulate production of intracellular trehalose can
also be effected by, for example, inhibiting the rate of
degradation of trehalose, expressing recombinant genes and inducing
uptake of exogenous trehalose.
[0032] "Ambient" is a term of art referring to the atmospheric
pressure or humidity or temperature of the room in which the
methods described are being performed. Ambient temperature is also
referred to as room temperature and is generally from about
15-25.degree. C.
[0033] "Residual moisture" is the amount of water remaining
(expressed in weight percent) after drying prokaryotic cells by the
methods described herein. Residual moisture can be measured by
Karl/Fischer Coulometer, as discussed in more detail below.
[0034] "Glass" is a term well understood in the art, especially as
applied to carbohydrate glasses. For purposes of this invention,
"glass" refers to a non-crystalline, vitreous, solid physical state
achieved upon sufficient loss of water.
[0035] As used herein, "foamed glass matrix" (FGM) refers to a
carbohydrate-containing glass that contains bubbles dispersed in
the glass, resulting in a foam. For purposes of this invention, a
foamed glass matrix contains less than about 5% residual moisture,
preferably less than about 4% residual moisture, more preferably
less than about 2% residual moisture.
[0036] "High osmolarity" refers to excessive solute concentration
in growth media. "Excessive" solute concentration means that solute
concentration (generally salts) is above the level at which a cell
exists and/or grows in its native environment.
[0037] "Viability" is a term well understood in the art, and is
consonantly used herein to mean manifestations of a functioning
living organism, such as metabolism and cell division. Methods to
measure viability are known in the art and are described
herein.
[0038] The present invention encompasses methods of producing
stabilized prokaryotic cells and the cells produced thereby. These
methods comprise the steps of increasing intracellular trehalose,
preferably by culturing or incubating the prokaryotic cells under
conditions that increase intracellular trehalose concentration to
an amount effective to increase storage stability; mixing the
prokaryotic cells with a drying solution which contains a
stabilizing agent, preferably a non-reducing carbohydrate such as
trehalose; and drying the resulting mixture such that a glass is
produced having less than about 5% residual moisture.
Growing Prokaryotic Cells to Increase Intracellular Trehalose
Concentration
[0039] To practice the methods of this invention, prokaryotic cells
can be grown under conditions that increase intracellular trehalose
concentration. Intracellular trehalose can be measured using
standard methods in the art as described below. Any prokaryotic
cell, particularly bacteria, containing trehalose synthase genes,
whether endogenous or recombinant, should be capable of producing
intracellular trehalose.
[0040] Many types of prokaryotic cells are known to synthesize
trehalose. Examples of bacteria that contain the trehalose synthase
gene include, but are not limited to, Enterobacteriaceae, such as
Salmonella and Escherichia (e.g., S. typhimurium and E. coli);
halophilic and halotolerant bacteria, such as Ectothriorhodospira
(e.g., E. halochloris); micrococcocaceae, such as Micrococcus
(e.g., M. luteus); Rhizobium species such as R. japonicum and R.
leguminosarum by phaseoli; Cyanobacteria and Mycobacteria species
such as M. tuberculosis, M bovis, and M. smegmatis. An alignment of
trehalose synthases encoded by genes from a variety of organisms is
shown in FIG. 1. Several other bacteria have been shown to have
trehalose synthase genes all of which are highly homologous to the
E. coli gene. These bacteria include Pseudomonas putidae and
Aeromonas salmonicida.
[0041] Determining whether a particular bacteria species contains
trehalose synthase gene(s) can be accomplished by, for example,
searching available nucleic acid (and/or protein) databases for the
presence of sequences that encode (or that correspond to) consensus
regions of the amino acid sequence for trehalose synthase genes.
Bacteria have two genes involved in trehalose synthesis (i.e.,
T-Phosphate synthase and T-6-P phosphatase), whereas yeast have at
least three genes. Generally, searching with probes specific for
the yeast genes also identifies the bacterial genes, albeit with
lower homology scores. Amino acid sequence alignments of trehalose
synthase show homology between bacteria, yeast and fungi and more
specific search and screening probes can be determined from these
alignments (FIG. 1). Alternatively, Southern blots can be produced
of genomic DNA from a test cell probed with DNA encoding all or a
functional portion of trehalose synthase gene. FIG. 2 shows a
Southern blot of the trehalose synthase genes of E. coli, and two
strains of Salmonella. Alternatively, PCR-based detection can be
used. These methods are well-known in the art.
[0042] Increases in intracellular trehalose can be obtained by
culturing the cells under stressful conditions, e.g., osmotic
shock, heat or oxygen limitation (shock), carbon/nitrogen
starvation, or any combination of the above. Alternatively, use of
inhibitors of enzyme(s) involved in trehalose degradation (i.e.,
trehalase), such as validomycin, also results in accumulation of
intracellular trehalose. Suitable conditions can be determined
empirically and are well within the skill of one in the art. While
not wishing to be bound to a particular theory, induction of
trehalose production under stressful conditions may trigger
synthesis or accumulation of other molecules beneficial for
preservation, such as betaine and chaperonins.
[0043] For bacteria, particularly Escherichia, trehalose production
can be stimulated by growing the cell(s) in conditions of high
osmolarity, i.e., solute (generally salt) concentrations sufficient
to stimulate trehalose production. Thus, the invention encompasses
culturing prokaryotic cells in osmolarity of at least about 350
mOsmoles to about 1.5 Osmoles, preferably at least about 400
mOsmoles to 1 Osmole, more preferably 250 mOsmoles to 500 mOsmoles.
The invention also encompasses culturing prokaryotic cells in
osmolarity of at least about 300 mOsmoles, preferably at least
about 400 mOsmoles, more preferably at least about 500 mOsmoles.
Generally, a minimum salt concentration of about 200 mOsmoles is
required but an effective concentration can be derived empirically.
A single salt can be sufficient to stimulate trehalose production,
for example, 200 mM NaCl. KCl and CaCl.sub.2 also stimulate
intracellular trehalose production, indicating that intracellular
trehalose production is not dependent on the action used or the
concentration of chloride in the growth medium. When
(NH.sub.4).sub.2SO.sub.4 is used, however, only about one half of
the amount of trehalose is produced compared to that produced in
the presence of KCl, NaCl and CaCl.sub.2. A combination of salts
can also be used. In addition, when used to increase the osmolarity
of the medium, a non-penetrant solute such as sorbitol and/or
glucose can contribute to the stimulation of trehalose
accumulation.
[0044] Examples of salts that can be used to increase the
osmolarity include, but are not limited to, sodium phosphate
(Na.sub.2PO.sub.4); potassium phosphate (KH.sub.2PO.sub.4);
ammonium chloride (NH.sub.4Cl); sodium chloride (NaCl); magnesium
sulfate (MgSO.sub.4); calcium chloride (CaCl.sub.2); thiamine
hydrochloride or any combination thereof. In a preferred
embodiment, minimal medium contains about 0.5 M salt. Even more
preferably, the 0.5 M salt is composed of the following:
Na.sub.2HPO.sub.4, 6 g/l; KH.sub.2PO.sub.4, 3 g/l; NH.sub.4Cl,
0.267 g/l; NaCl, 29.22 g/l; 1 M MgSO.sub.4, 1 ml/l; 0.1 M
CaCl.sub.2 (1 ml/l) thiamine HCl, 1 ml/l; with glucose at final
concentration of 2.5% w/v. Sufficient glucose should be available
for a carbon source and trehalose production. Determining
sufficient glucose concentrations can be determined empirically and
is well within the skill of one in the art.
[0045] The salt concentration (i.e., osmolarity) required to
stimulate and/or induce trehalose production will depend upon the
genus, species, and/or strain of the prokaryotic cell used.
Preferably, cell(s) are grown in a minimal medium containing salt.
Commercially available minimal medium is supplemented with desired
salts and/or other solutes, although minimal medium is not
essential and defined media can also be used. The time required to
initiate and achieve the desired level of intracellular trehalose
concentration will vary depending on the level of osmolarity as
well as the genus, species and/or strain of prokaryotic cell used
and can be determined empirically. Trehalose synthesis will
generally begin within an hour of placing cells in condition
designed to stimulate trehalose production. Generally, in E. coli
the amount of intracellular trehalose reaches a maximum at about 15
to 20 hours after placing cells in conditions that stimulate
trehalose production.
[0046] To induce intracellular trehalose production by osmotic
shock, the total concentration of salt(s) in the medium should be
at least about 0.2 M, preferably at least about 0.4 M, more
preferably at least about 0.5 M. In the case of E. coli, the total
concentration of salt(s) should not exceed 0.6 M. At about 0.6 M or
above, intracellular trehalose production declines in E. coli. The
salt concentration required for the desired result may vary
depending on the general/species/strain used, and can be determined
empirically.
[0047] Intracellular trehalose can also be increased using
recombinant methods which are well known in the art. For instance,
prokaryotic cells can be transfected with a DNA plasmid comprising
a DNA sequence encoding an appropriate trehalose synthase gene.
Suitable genes are available from a wide variety of resources as
indicated by the number of genes depicted in FIG. 1 and other genes
recently identified. The gene in turn is operatively linked to a
suitable promoter, which can be constitutive or inducible.
Recombinant methods are described in a variety of references, such
as "Molecular Cloning: A Laboratory Manual," second edition
(Sambrook et al., 1989).
[0048] Intracellular trehalose can be measured by using assays
known in the art, such as by high pressure liquid chromatography
(HPLC), coupled with electrochemical detection and glucose assay
(Trinder assay using trehalase) for quantitative enzymatic
determination of trehalose. Thin layer chromatography can be used
as a qualitative method for the separation of different
carbohydrates. Refractive index detection provides another means of
detecting sugars quantitatively.
[0049] In measuring trehalose by HPLC, cells are disrupted and
intracellular trehalose preferentially solubilized in 70% ethanol,
followed by removing triglycerides by chloroform extraction.
Intracellular trehalose concentration is determined by multiplying
trehalose concentration (as determined by a standard curve) by the
fraction of final volume of supernatant divided by pellet volume. A
more detailed description of this assay is provided in Example
1.
[0050] Preferably, the concentration of intracellular trehalose is
at least about 50 mM; more preferably, at least about 100 mM; more
preferably, at least about 150 mM; more preferably, at least about
200 mM; more preferably, at least about 250 mM; and even more
preferably, at least about 300 mM. We have found that stability of
bacteria decreases markedly using the methods described herein if
the intracellular trehalose concentration is below about 30 mM.
Thus, the invention encompasses culturing the prokaryotic cells
under conditions that stimulate intracellular production of
trehalose, wherein intracellular concentration of trehalose reaches
at least about 30 mM, preferably at least about 50 mM, preferably
at least about 100 mM, more preferably at least about 150 mM, more
preferably at least about 200 mM, more preferably at least about
250 mM, and even more preferably at least about 300 mM.
[0051] The time required for stimulating intracellular trehalose
production depends, inter alia, on the nature of the prokaryotic
cells (including genus, species, and/or strain) and the conditions
under which trehalose induction occurs (i.e., whether by osmotic
shock, oxygen deprivation, etc.). For trehalose induction by
osmotic shock, the time required for maximum concentration of
intracellular trehalose in turn depends on the degree of osmolarity
as well as the particular salts used. For example, in E. coli,
ammonium sulfate ((NH.sub.4).sub.2SO.sub.4) stimulates about half
the amount of intracellular trehalose concentration as NaCl,
CaCl.sub.2 or KCl. For E. coli in 0.5 M salt minimal media, maximum
intracellular trehalose concentration occurs within about 10-17
hours, with significant induction by 17 hours after osmotic shock
(Example 1; FIG. 6).
[0052] As is readily apparent to those skilled in the art,
achieving a desired intracellular trehalose concentration can also
be effected by other means such as introducing trehalose into the
cell(s). This can be accomplished, for example, by culturing cells
in the presence of trehalose while subjecting the cell(s) to
conditions that permeabilize the cell wall and membrane. Examples
of such conditions include, but are not limited to, conditions that
effect membrane phase transition (such as cycles of cooling and
warming or osmotic shock) and electroporation. The intracellular
trehalose concentration can be determined for these conditions as
described above. Conditions that effect membrane phase transition
especially apply to Gram negative bacteria.
[0053] Accordingly, one embodiment of the present invention is a
method of preserving prokaryotic cells comprising the steps of
culturing the prokaryotic cells under conditions that increase
intracellular trehalose concentration to a level effective to
increase storage stability in the methods described herein, mixing
the prokaryotic cells with a drying solution which contains a
stabilizing agent, and drying the prokaryotic cells such that a
glass is produced having less than about 5% residual moisture.
[0054] Mixing the prokaryotic cells with drying solution. After
intracellular trehalose is increased to the desired degree, the
prokaryotic cells are harvested by, for instance, centrifugation
and resuspended in a drying solution containing a stabilizing
agent, preferably a non-reducing carbohydrate such as
trehalose.
[0055] Particularly preferred non-reducing carbohydrates are
trehalose, maltitol (4-O-.beta.-D-glucopyranosyl-D-glucitol),
lactitol (4-O.beta.-D-galactopyranosyl-D-glucitol), palatinit [a
mixture of GPS (.alpha.-D-glucopyranosyl-1.fwdarw.6-sorbitol) and
GPM (.alpha.-D-glucopyranosyl-1.fwdarw.6-mannitol)], and its
individual sugar alcohol components GPS and GPM and hydrogenated
maltooligosaccharides and maltooligosaccharides.
[0056] In addition to trehalose, suitable stabilizing agents
include, but are not limited to, non-reducing glycosides of
polyhydroxy compounds selected from sugar alcohols and other
straight chain polyalcohols. Other useful stabilizing agents
include neotrehalose, lactoneotrehalose, galactosyl-trehalose,
sucrose, lactosucrose, raffinose, stachyose and melezitose.
Carbohydrates with mild reducing activity, such as maltohexose,
maltoheptulose, Sepharose and Dextran, can also be used with
Maillard reaction inhibitors as described in patent application
PCT/GB95/01967. Maillard reaction inhibitors can also be used to
improve the performance of unstable reducing carbohydrates such as
sucrose.
[0057] The concentration of non-reducing carbohydrate(s) in the
drying solution will depend on several variables, most particularly
the genus, species, and/or strain of prokaryotic cell that is being
stabilized and the method of drying. For E. coli, the non-reducing
carbohydrate (trehalose) concentration is preferably at least about
25%, more preferably at least about 35%, even more preferably at
least about 45% (w/v). Preferably, the carbohydrate concentration
should be less than about 50%, as higher concentrations may
interfere with effective drying.
[0058] The solvent(s) that forms the basis for the drying solution
can be any of a number of substances, provided it does not
significantly affect cell viability. Preferably, the solvent is
aqueous.
[0059] The drying solution can optionally contain additives that
contribute to overall stability of the prokaryotic cells.
Generally, preferred additives increase the viscosity of the drying
solution, which in turn enhances the drying process by more
efficient foam production with higher T.sub.gs. Examples of
additives include, but are not limited to, polyvinylpyrollidone
(Kollidon Series: 12, 17, 25, 30, 90; BASF), carboxymethyl
cellulose (Blanose HF; Aqualon) hydroxypropyl cellulose and
hydroxyethyl starch (HES; MW 200,000). Preferably, Kollidon 90 is
present in the drying solution at a concentration of about 1.5%.
Preferably, the concentration of carboxymethyl cellulose is about
0.1%. Particularly preferred is a drying solution containing about
45% trehalose and either about 1.5% Kollidon 90 or about 0.1%
carboxymethyl cellulose. Other additives that can be used include
volatile salts, which contribute to effective drying (via foam
formation). Examples of volatile salts include, but are not limited
to, ammonium bicarbonate, ammonium chloride, ammonium acetate and
ammonium sulfate. However, when using these salts, it is possible
that more effective drying may be counteracted by lower viability
due to pH and salt-specific effects.
[0060] The volume of the drying solution added to the prokaryotic
cells, and thus the density of the prokaryotic cells in the drying
solution, can vary. However, too low a cell density proportionately
increases the drying time per cell; too high a density may
adversely affect rapidity and/or efficiency of foam formation and
thus drying. Moreover, too high a cell density could result in
higher concentration of anti-foaming agents produced by the cells.
Preferably, the cell density is about 4 to 8.times.10.sup.9 cells
(CFU) per ml, although densities as high as 2.times.10.sup.10 cells
per ml have been used with success. Generally, the volume of drying
solution is significantly less than the volume of culture medium
used for increasing intracellular trehalose concentration. The
optional volume will vary somewhat on the types of cells and
solutes and can be readily determined empirically.
[0061] Drying the prokaryotic cells. Upon suspending in the drying
solution, the prokaryotic cells are then dried such that a glass is
formed. Drying can be effected using methods known in the art,
including, but not limited to, air (i.e., ambient temperature)
drying, spray drying, and freeze drying. As used herein, the glass
containing the dried prokaryotic cells preferably has a residual
moisture content less than about 5%.
[0062] Drying is preferably performed at pressure less than ambient
(i.e., vacuum). Preferably, the pressure is about 0.1 to 0.075
Torr/mm Hg. More preferably, the pressure is about 0.075 to 0.05
Torr/mm Hg. Most preferably, the pressure is about 0.05 to 0.03
Torr/mm Hg and external temperature is about 40.degree. C.
[0063] Preferably, drying occurs above freezing temperatures and
under a vacuum such that a foamed glass matrix (FGM) is formed.
PCT/GB96/0136. Vacuum drying under freezing conditions will lead to
lower viability. For creation of a vacuum, any vacuum drier with a
control, preferably programmable control, of the vacuum pressure
and external temperature can be used. As an example, a pump is
capable of providing a vacuum of 0.01 Torr/mm Hg and evacuating the
product chamber down to 0.2-0.01 Torr/mm Hg in 15-20 minutes. The
machines used in the present work were the FTS Systems Inc. (Stone
Ridge, N.Y.) Model TDS 0007-A with a VP-62P vacuum pump and a
FD-0005-A condenser module or the Labconco, Inc. (Kansas City)
Model No. 77560 with a Lyph-Lock 12 condenser unit and an Edwards
E2M8 two-stage vacuum pump.
[0064] Reduction of the external pressure has at least two
desirable effects. First, it reduces the vapor pressure of the
solvent in the gas phase, thus accelerating evaporation and drying.
The increased rate of evaporation causes evaporative cooling unless
external heat is applied to replace the latent heat of evaporation.
Under vacuum, the rate of drying is limited by this energy input.
Thus, the effect of increasing the external temperature is,
surprisingly, to accelerate the rate of drying and not to increase
the sample temperature. The second effect of reduced external
pressure is to drastically lower the boiling point of the sample.
Boiling can therefore be conducted by a very modest rise in sample
temperature which does not have a deleterious effect on the
product.
[0065] Preferably, drying occurs in two stages: first, holding
external temperature constant for a period of time; and second,
increasing the external temperature until drying is complete. The
temperature can be increased gradually, for example, 10 degrees
over an hour, or, more preferably the temperature can be increased
in equal increments, with each increment held constant for a period
of time. In one embodiment, the temperature is maintained at about
40.degree. C. for about 16 hours, followed by gradually increasing
the temperature to about 80.degree. C. over about the next 4
hours.
[0066] In a preferred embodiment, the prokaryotic cells are dried
as follows: the pressure is adjusted to 30 mT, with initial shelf
temperature of 40.degree. C. for 16 hours; followed by
incrementally increasing the shelf temperature to 80.degree. C. at
a rate of 2.5.degree. C. per minute in increments of 2.degree. C.,
while holding each increment for about 12 minutes. Following this
protocol, foaming typically occurs within 60 minutes of the
initiation of drying, and the drying procedure is completed within
24 hours without substantially compromising viability. Example 6
provides a protocol.
[0067] FGMs are also formed by evaporating bulk solvent from the
drying solution to obtain a syrup, exposing the syrup to a pressure
and temperature sufficient to cause boiling or foaming of the
syrup, and removing moisture so that residual moisture does not
exceed about 4%, preferably about 3%, more preferably about
2.5%.
[0068] In the primary drying step, the solvent is evaporated to
obtain a syrup. Typically, a "syrup" is defined as a solution with
a viscosity in the region of 10.sup.6-10.sup.7 Pascal seconds. The
syrup is not defined as a fixed concentration, but is a result of
the bulk of the solvent evaporating from the mixture. Typically, a
syrup is a viscous mixture containing the glass matrix-forming
material and/or additives and/or prokaryotic cells, in a
significantly higher concentration than that of the initial
mixture. Typically, the evaporation step is conducted under
conditions sufficient to remove about 20% to 90% of the solvent to
obtain a syrup. The viscosity of the syrup is preferably such that
when the syrup boils, evaporation from the increased surface area,
provided by extensive bubble formation, results in its
vitrification.
[0069] Under the vacuum, rapid drying continues until the viscosity
of the sample begins to increase. At this point, the reduced
mobility of water molecules through the viscous syrup reduces the
rate of evaporative cooling and the sample temperature rises until
it reaches the boiling point at the reduced pressure. On boiling, a
large increase in the area of the liquid/gas interface occurs due
to the bubbling of the syrup. This increased evaporative surface
causes a sharp increase in the drying rate and the liquid foam
dries into solid glass foam (FGM). Typically, this occurs soon
after boiling.
[0070] Temperatures for the boiling step can be above or below
ambient temperature. Preferably, the external temperature for the
boiling step is about 5 to 80.degree. C. More preferably, the
external temperature is about 5 to 60.degree. C.; even more
preferably, about 5 to 35.degree. C.
[0071] The drying process results in formation of bubbles which
greatly increases the evaporative surface area of the syrup. This
allows increased evaporation of residual solvent and the FGM
vitrifies as a solid foam of the bubbles which result from the
boiling step. The endpoint of the boiling step can be determined by
an increase in sample temperature; which is preferably maintained
for a period of time sufficient to ensure complete drying. The
optimum time varies from sample to sample but is easily
determinable by one of skill in the art.
[0072] Various container shapes and sizes can be processed
simultaneously. Ideally, the container size used is sufficient to
contain the initial mixture and accommodate the volume of the dried
cells formed thereof. Generally, 3 ml pharmaceutical vials are
used. Any such vials can be used, including Wheaton molded and
tube-cut vials. Preferably, the vials are moisture resistant so as
to eliminate any deleterious effects due to moisture uptake by a
sample.
[0073] Residual moisture content can be measured using assays known
in the art, such as Karl Fischer coulometric method and gravimetric
method. For determination of residual moisture using a Coulometer,
residual moisture is extracted using formamide, followed by
measurement using a Coulometer. Percent moisture in the sample
(w/w) is determined using the following formula: 1 test sample -
blank .times. 10 3 .times. 10 2 wt of dried sample ( mg ) .times.
10 2 .times. 10 3 % residual moisture
[0074] A more detailed description of this assay is provided in
Example 2. Preferably, residual moisture will be equal to or less
than about 5%, more preferably less than about 4%, more preferably
equal to or less than about 3% even more preferably equal to or
less than about 2.5%. When cells are dried more rapidly by
gradually increasing the temperature, as described above, residual
moisture may drop below 2%. The allowable maximum for different
cell types can easily be determined empirically. Generally,
residual moisture above about 5% can be detrimental to viability.
This varies depending, inter alia, on the genus/species/strain
used, the concentration and type of non-reducing carbohydrate used
in the drying solution, method of drying and type of storage.
[0075] The resultant glass or FGM containing the dried, stabilized
prokaryotic cells should have a T.sub.g sufficiently high to
preserve the cells. "T.sub.g" refers to the temperature at which
the glass undergoes a transition into liquid phase. Variables that
determine T.sub.g include, but are not limited to, the amount of
residual moisture of the dried preparation(s) and the type of
stabilizing agent used. Generally, protein and polysaccharides
raise T.sub.g, while salts generally lower T.sub.g. FIG. 4
illustrates the relationship between T.sub.g and percent residual
moisture.
[0076] For purposes of this invention, T.sub.g should be at least
about 70.degree. C., preferably at least about 75.degree. C., more
preferably at least about 80.degree. C., even more preferably at
least about 85.degree. C., most preferably at least about
90.degree. C. T.sub.g can be determined using standard techniques
in the art, such as differential scanning calorimetry. Generally,
the higher the T.sub.g, the higher the allowable storage
temperature.
[0077] The length of time required to achieve the desired residual
moisture and/or T.sub.g will depend on several variables,
including, but not limited to, sample size, pressure and
temperature. Generally, the longer the samples are dried, the lower
the residual moisture (and hence the greater the T.sub.g). FIG. 5
shows the relationship between residual moisture and length of
drying time. Drying can be achieved in as few as 20 hours, more
generally within about 24 hours. Gradually increasing the
temperature during drying, as described above, lowers the drying
time without significantly reducing cell viability (Example 6).
[0078] Prokaryotic cells dried by the methods disclosed herein can
be stored for varying lengths of time at ambient or higher
temperatures. The length of time the dried, stabilized prokaryotic
cells can be stored will depend, inter alia, on the genus, species,
and/or strain of the prokaryotic cell, the degree of intracellular
trehalose production and/or concentration, the concentration and
type of stabilizing agent in the drying solution, the drying
protocol followed, the amount of residual moisture after drying,
and the acceptable degree of viability.
[0079] Reconstitution of stabilized cells. The prokaryotic cells
can be reconstituted after drying by adding a suitable solvent.
Thus, the invention includes methods of reconstituting prokaryotic
cells that have been obtained by the methods described herein. The
nature and amount of solvent used for reconstitution will depend
upon the prokaryotic cells as well as their intended use. Such
determinations can be made empirically by those skilled in the art.
Generally, cells can be reconstituted with an aqueous solvent. If
the cells are to be used as a pharmaceutical, reconstitution is
preferably with a sterile physiologically acceptable buffer.
[0080] If the prokaryotic cells are to be used as a vaccine, and
thus as an immunogenic agent, an adjuvant can be added in an amount
sufficient to enhance the immune response to the immunogen. The
adjuvant can be added to the prokaryotic cells before drying, for
example, cholera B toxin subunit can be dried simultaneously with
V. cholera. Alternatively the adjuvant can be separately
reconstituted along with the prokaryotic cells.
[0081] Suitable adjuvants include, but are not limited to, aluminum
hydroxide, alum, QS-21 (U.S. Pat. No. 5,057,540), DHEA (U.S. Pat.
Nos. 5,407,684 and 5,077,284) and its derivatives (including salts)
and precursors (e.g., DHEA-S), beta-2 microglobulin (WO 91/16924),
muramyl dipeptides, muramyl tripeptides (U.S. Pat. No. 5,171,568),
monophosphoryl lipid A (U.S. Pat. No. 4,436,728; WO 92/16231) and
its derivatives (e.g., Detox.TM.), and BCG (U.S. Pat. No.
4,726,947). Other suitable adjuvants include, but are not limited
to, aluminum salts, squalene mixtures (SAF-1), muramyl peptide,
saponin derivatives, mycobacterium wall preparations, mycolic acid
derivatives, nonionic block copolymer surfactants, Quil A, cholera
toxin B subunit, polyphosphazene and derivatives, and
immunostimulating complexes (ISCOMs) such as those described by
Takahashi et al. (1990) Nature 344:873-875.
[0082] For veterinary use and for production of antibodies in
animals, mitogenic components of Freund's adjuvant can be used. The
choice of an adjuvant depends in part on the stability of the
vaccine in the presence of the adjuvant, the route of
administration, and the regulatory acceptability of the adjuvant,
particularly when intended for human use. For instance, alum is
approved by the United States Food and Drug Administration (FDA)
for use as an adjuvant in humans.
[0083] Cell viability (i.e., survival) can be determined using any
of a number of techniques known in the art, such as, for example, a
plate assay for colony forming units (CFU). Viability can be
determined at any time, including before and immediately after the
cells are dried as well as upon various times during storage. It
may be desirable to test viability after reconstitution but before
application and/or administration of the cells.
[0084] For a plate assay, cells are reconstituted at desired
time(s) with a desired solvent, generally sterile distilled water
of a volume at least equal to the volume of the dried cells. After
vortexing, solutions of reconstituted cultures are diluted
(generally 10-fold) in mineral media (for example M9 minus a carbon
source) and plated in triplicate on appropriate nutrient again
within 30 minutes, more preferably within 15 minutes. After
incubation at 37.degree. C. for 18-24 hours, the number of colony
forming units (CFU) is determined. Survival is calculated as a
percentage of zero time colony counts. A more detailed description
of the plate viability assay is provided in Example 2.
[0085] Compositions of cells made by the methods herein. The
invention also encompasses compositions comprising prokaryotic
cells obtained by the methods described herein. The compositions
include, but are not limited to, dried prokaryotic cells and
reconstituted prokaryotic cells made according to the methods
described herein. The compositions may further comprise any
pharmaceutically acceptable vehicle or excipient, which are well
known in the art.
[0086] The following examples are provided to illustrate but not
limit the invention. S. typhimurium 1344 and S. typhi Ty21a were
obtained from the National Institute of Biological Standards and
Control, South Mimms, UK.
EXAMPLE 1
Effect of Osmotic Shock on Production of Intracellular Trehalose in
E. coli
[0087] E. coli NCIMB strain 9484 was cultured in Evans medium (pH
7.0; Table 1) containing one of a variety of agents for increasing
osmotic pressure. After overnight incubation at 37.degree. C. in
initial Evans medium, a 4 ml culture of E. coli grown in Evans
medium under nitrogen limitation was used to inoculate a 200 ml
culture of Evans medium osmotic shock.
1TABLE 1 Evans medium and Evans osmotic shock medium Osmotic Shock
Evans Initial Evans Medium medium glucose 140 mM 10 g/l glucose
NH.sub.4Cl 5 mM 3-5 g/l NH.sub.4Cl (15 mM) KCl 5 mM 0.5 M NaCl =
29.22 g/l Na.sub.2SO.sub.4 1.8 mM 1.8 mM citric acid 1 mM 1 mM
MgCl.sub.2 0.3 mM 0.3 mM CaCl.sub.2 0.5 mM 0.5 mM NaH.sub.2PO.sub.4
5.6 mM 5.6 mM Na.sub.2HPO.sub.4 20 mM 20 mM ZnSO.sub.4 3.8 mM 3.8
mM FeCl.sub.2 50 mM 50 mM MnCl.sub.2 25 mM 25 mM CuCl.sub.2 2.5 mM
2.5 mM H.sub.3BO.sub.3 2.5 mM 2.5 mM CoCl.sub.2 0.5 mM 0.5 mM
chloramphenicol 50 mg/liter 50 mg/liter
[0088] Intracellular trehalose concentration was measured as
described below at various times after the initiation of osmotic
shock.
Determination of Intracellular Trehalose Concentration
[0089] Intracellular concentration of trehalose was determined
using high pressure liquid chromatography (HPLC) as follows.
Trehalose standards were prepared by first making 10 mM trehalose
in 70% ethanol, followed by 10-fold serial dilution from 10 mM to
10 nM using 70% ethanol as diluent. Thirty .mu.l of the standard
was placed in a microtube which was placed in an 80.degree. C.
water bath for 5 minutes, while noting the initial volume of the
supernatant following incubation. Microtubes were centrifuged at
13,000 rpm for 10 minutes and the supernatant removed. After adding
an equal volume of chloroform to the supernatant, the samples were
vortexed and centrifuged at 13,000 rpm for 10 minutes. The
chloroform extractions were repeated another two times. The final
volume of the supernatant was adjusted to 500 .mu.l using deionized
water. A calibration curve was generated by testing samples at
varying concentrations.
[0090] Cell samples were prepared for analysis by disrupting the
cell wall by sonication (any other method such as mortar and
pestle, osmotic lysis, beads can be used) coupled with the
preferential solubilization of trehalose in 70% ethanol, followed
by removing triglycerides by chloroform extraction. One ml of cell
suspension was aliquoted into a microtube, which was centrifuged at
13,000 rpm for 10 minutes. The pellet was resuspended with 100
.mu.l of 70% ethanol (initial volume). The pellet volume was
determined by measuring the relative increase in the initial volume
following resuspension of the cells. The cell suspension was
incubated in a water bath at 80.degree. C. for 5 minutes. The tubes
were centrifuged at 13,000 rpm for 10 minutes and the supernatant
removed. An equal volume of chloroform was added and the
centrifugation step repeated. Chloroform extraction was performed a
total of three times. The final volume of supernatant was adjusted
to 500 .mu.l using deionized water.
[0091] Quantitation of trehalose was achieved by HPLC (Beckman
Instruments), using a Dionex CarboPac PA 100 analytical column,
with a Dionex ED40 pulsed amperometric electrochemical detector.
Total trehalose concentration from the original cell pellet was
determined as a fraction of the final volume extracted and the
pellet volume multiplied by the trehalose concentration determined
using the following formula: 2 Final volume of supernatant Pellet
volume .times. Trehalose concentration
[0092] Final volume of supernatant was the aqueous volume remaining
after the final chloroform extraction. Pellet volume was the
difference in the resuspended pellet following the addition of 100
.mu.l of 70% ethanol. Concentration of trehalose formed was
determined using the trehalose concentration curve.
[0093] The results obtained are shown in FIG. 6. Significant
increases in intracellular trehalose concentrations were observed
at 15-17 hours after initiation of osmotic shock, with values
peaking at less than 20 hours.
EXAMPLE 2
Stabilization and Reconstitution of E. coli using Trehalose
[0094] E. coli (strain 9484) was placed in 100 ml batch cultures of
a minimal medium related to M9 (minimal medium) but with high (0.5
M) salt content (Na.sub.2HPO.sub.4, 6 g/l; KH.sub.2PO.sub.4, 3 g/l;
NH.sub.4Cl, 0.267 g/l; NaCl, 29.22 g/l; 1 M MgSO.sub.4, 1 ml/l; 0.1
M CaCl.sub.2, 1 ml/l; thiamine HCl, 1 ml/l; glucose at final
concentration of 2.5% w/v). This is "modified M9 medium." Cells
were grown for 22 hours at 37.degree. C. with shaking. A control
culture where the medium was supplemented with 20 mM betaine, in
which trehalose synthesis would be markedly reduced, was also
prepared. Samples of cultures were removed for trehalose
determination (3.times.1 ml) as described in Example 1 and protein
estimation by the Bradford assay (3.times.10 ml; Bio-Rad).
[0095] Two 25 ml aliquots of the test and control culture were
harvested by centrifugation at 10,000 rpm for 10 minutes. Cell
pellets were resuspended in 5 ml of 45% trehalose, 1.5%
polyvinylpyrollidone (Kollidon 90; BASF) or 0.1%
carboxymethylcellulose (Blanoes HF; Aqualon). The suspensions were
then pooled to a total volume of 10 ml with a typical density of
4-8.times.10.sup.9 bacteria/ml and 300 .mu.l aliquots dispensed
into 3 ml pharmaceutical vials.
[0096] Bacteria were dried under vacuum without freezing in a
modified FTS freeze dryer according to the following protocol:
vacuum, 30 mT; initial shelf temperature 40.degree. C. for 16
hours, followed by ramping to 80.degree. C. at a rate of
2.5.degree. C. per minute in increments of 2.degree. C. with a
holding time of 12 minutes per increment. Foaming occurred within
approximately 60 minutes of initial drying.
[0097] Residual moisture content was determined as follows. One ml
of formamide was carefully dispensed into each vial containing the
dried bacteria in trehalose. One ml of formamide added to an empty
vial served as a control. Residual moisture was extracted by mixing
for 15 to 20 minutes at room temperature. For the analysis, 100
.mu.l of the blank (control) formamide was added to a reaction
vessel using disposable needles and syringes, and the value
registered by the Coulometer (Karl/Fischer) was recorded. Care was
taken not to introduce air into the formamide samples, as air
contains water vapor. The test (and control) samples were measured
in duplicate. The value determined by the Coulometer was equal to
.mu.g of water. Test sample less blank divided by 100 is equal to
.mu.g of water per .mu.l of formamide in the sample. Percent
moisture in the dried sample (w/w) is: 3 test sample - blank
.times. 10 3 .times. 10 2 wt of dried sample ( mg ) .times. 10 2
.times. 10 3 %
[0098] Viability was determined immediately after completion and at
various times during storage at 37.degree. C. using a plate assay.
For the plate assay, serial 10-fold dilutions of cells were set up
by using minimal medium minus a carbon source as a sterile
diluent.
[0099] Thirty .mu.l of the cell suspension from the sixth dilution
tube was added to each of 3 LB (Luria-Bertuni) plates, using a
sterile glass spreader to spread the culture over the entire
surface of the plate. The plates were incubated overnight at
37.degree. C., and the colonies counted.
X=number of colonies X.times.33-1/3.times.1.times.10.sup.6 dilution
section CFU/ml
[0100] The results for storage at 37.degree. C. up to 45 days are
shown in FIG. 3. Greater than 50% viability (typically 50-80%) in
the trehalose induced cells was observed in samples reconstituted
immediately after drying. More significantly, no further losses in
viable cell recovery were observed on storage of the dried cells,
even after 45 days storage at 37.degree. C. (FIG. 3.).
EXAMPLE 3
Southern Blot Analysis to Detect Presence of Trehalose Synthase
Gene
[0101] DNA was prepared from E. coli, S. typhimurium 1344 (1344),
and Salmonella typhi Ty21a (Ty21a) using standard methods. E. coli
and Salmonella genomic DNA were digested with restriction
endonucleases Hind III (H), EcoRI (R), or Bam H1 (B), separated on
a 0.8% TBE (Tris-borate electrophoresis buffer) agarose gel and
blotted onto nylon filters. The filters were screened using a
.sup.32P-labeled probe corresponding to the otsA/B region of E.
coli that codes for the trehalose synthase genes in E. coli. After
hybridization, the filters were washed at low stringency. Exposure
of the gels to X-Ray film was overnight for E. coli and three days
for Salmonella spp.
[0102] The presence of trehalose synthase genes was detected in
both strains of Salmonella as shown in FIG. 2. Fainter bands were
detected when filters were washed under higher stringency
conditions.
EXAMPLE 4
Induction of Trehalose Synthesis in Salmonella
[0103] Salmonella typhimurium (1344) was grown overnight at
37.degree. C. in either M9 (minimal) medium with and without 0.5 M
NaCl. Cells were harvested by centrifugation and analyzed for
intracellular trehalose concentration by HPLC analysis as described
in Example 1. The results are shown in FIG. 7. Growth in high salt
medium showed at 4 to 5 fold induction of trehalose synthesis.
EXAMPLE 5
Relation Between Tg and Residual Moisture
[0104] E. coli (strain 9484)were grown in M9 media containing high
salt as described in Example 2. For drying, cells were suspended in
an aqueous drying solution containing 45% trehalose and 1.5%
Kollidon 90 and dried for 3-24 hrs under vacuum as described in
Example 2. Cells were collected at various times, and the residual
water content and Tg were measured on aliquots of the same sample
to eliminate any possible vial-to-vial variation. The results of
the relationship of Tg and residual moisture are shown in FIG.
4.
EXAMPLE 6
Comparison of Effect of Slower and Faster Drying on Viability
[0105] E. coli (strain 9484)were grown in modified M9 media
described in Example 2. For drying, cells were suspended in an
aqueous drying solution containing 45% trehalose and 0.1%
carboxymethyl cellulose (Blanose H. F., Aqualon).
[0106] Two different drying protocols were followed: (a) pressure,
30 mT; external temperature 40.degree. C. for 16 hours, followed by
increasing (ramping) the temperature to 80.degree. C. at the rate
of 0.04.degree. C./minute in increments of 2.degree. C., holding
each increment for about 60 minutes (slow drying); (b) pressure, 30
mT; external temperature 40.degree. C. for 16 hours, followed by
increasing the temperature to 80.degree. C. at the rate of
2.5.degree. C./minute in increments of 2.degree. C., holding each
increment for about 12 minutes (fast drying).
[0107] Viability was measured immediately after drying. The samples
prepared by fast drying were no less viable than those samples
prepared by slow drying. Ranges between about 48% and 52% were
observed for the "fast" dried samples, while between about 40% and
52% were observed for the "slow" dried samples. On average, the
"fast" dried samples displayed higher viability than the "slow"
dried samples.
[0108] The effect of length of drying time on viability is shown in
FIG. 8. The drying solution contained 45% trehalose and 0.1% CMC;
the FTS drying protocol was 30 mT ST 40.degree. C. for varying
times.
EXAMPLE 7
Comparison of the Effects of Different Excipients on Stabilising
the Outer Membrane of E. coli 9894 Following Intracellular
Induction of Trehalose
[0109] E. coli strain 9894 was inoculated in 100 ml batch cultures
of minimal medium related to M9 but with high (0.5M) salt content
as described in Example 2. Cells were grown for 22 hours at
37.degree. C. in a shaking incubator (early stationary phase).
Samples of cultures were removed for trehalose determination
(3.times.1 ml) and protein estimation by the Bradford assay
(3.times.10 ml; Bio-Rad). Trehalose concentration was expressed as
.mu.mol (mg protein).sup.-.
[0110] Intracellular concentration of trehalose was determined
using ion exchange chromatography with electrochemical detection.
Calibration standards were prepared by first making a stock
solution of 1 mM trehalose, glucose, sucrose and maltose standards
in water, followed by serial dilutions from 1 mM to 2.5 .mu.M using
water as a diluent.
[0111] One ml of cell suspension was aliquoted into a microtube,
which was centrifiged at 13,000 rpm for 10 minutes and the
supernatant removed. The cell pellet was resuspended in 200 .mu.l
of 80% ethanol. The cell suspension was prepared for analysis by
disrupting the cell wall in a 80.degree. C. bath for 10 minutes,
coupled with preferential solubilisation of all intracellular
sugars in 80% ethanol. The suspension was centrifuged and the
supernatant removed. An equal volume of chloroform was added to the
supernatant and vortexed, and the sample was centrifuged removing
triglycerides by the chloroform extraction. The aqueous layer was
transferred into a fresh Eppendorf tube and the chloroform
extraction repeated. The aqueous layer was aliquotted into HPLC
vials and vacuum dried, followed by rehydration using 500 .mu.l
sterile water.
[0112] Quantitation of trehalose was achieved by HPLC (Dionex
DX-500), using a Dionex CarboPac PA analytical column, with a
Dionex ED40 pulsed amperometric electrochemical detector. The
concentration of trehalose was determined from the calibration
curve.
[0113] Two 30 ml aliquots from each flask were harvested by
centrifugation at 10,000 rpm for 10 minutes. Cell pellets were
resuspended in 8 ml of 25-45% sugar, 0.1% CMC (sodium carboxymethyl
cellulose; Blanose 7HF; Aqualon). The suspensions were then pooled
to a total volume of 16 ml with a typical cell density of
4-8.times.10.sup.9 CFU/ml and 300 .mu.l aliquots dispensed into 3
ml pharmaceutical vials.
[0114] Bacteria were dried under vacuum without freezing using the
following protocol: vacuum, 30 mT; initial shelf temperature
40.degree. C. for 16 hours, followed by ramping to 80.degree. C. at
a rate of 2.5.degree. C./min in increments of 2.degree. C. with a
holding time of 12 minutes per increment. Foaming occurred between
60-120 minutes of initial drying.
[0115] Viability was determined immediately before and after the
completion of the drying procedure and at various times during
storage at 37.degree. C. using a plate assay as described in
Example 2. The residual moisture content and the glass transition
temperature were also determined.
[0116] The results for storage at 37.degree. C. are shown in Table
2. No significant loss in viable cell recovery was observed after 6
weeks storage of E. coli at 37.degree. C. using the non-reducing
sugars trehalose, palatinit or lactitol as excipients for
stabilizing the outer membrane. More significantly, greater than
99% loss was observed for the reducing sugar glucose.
2TABLE 2 E. coli 9484 viable cell recovery immediately after
completion of Q-T4 drying and following 3 and 6 weeks storage at
37.degree. C. % Viable Cell Recovery after storage at 37.degree. C.
Excipient Day 0 Week 3 Week 6 Trehalose 36 52 45 Palatinit 49 49 51
Lactitol 42 36 34 Glucose 0.8 0.1 0.05
EXAMPLE 8
Comparison of Q-T4 (The Method of Example 2) and Freeze-dried E.
coli
[0117] E. coli NCIMB strain 9484 was inoculated in 250 ml batch
culture of modified M9 medium. The composition of this medium was
described in Example 2. Cells were grown for 24 hours at 37.degree.
C. in a shaking incubator until early stationary phase. Samples of
cultures were removed for trehalose determination (6.times.1 ml)
and protein estimation by the Bradford assay (5.times.10 ml) as
described in Example 7.
[0118] Eight 25 ml aliquots were removed from the flask and the
bacteria harvested by centrifugation at 10,000 rpm for 10 minutes.
Cell pellets were resuspended in 8 ml of 45% trehalose, 0.1% CMC
(sodium carboxymethyl cellulose; Blanose 7HF; Aqualon). The cell
suspensions were then pooled to a total volume of 64 ml with a
typical cell density of 0.5-1.2.times.10.sup.9 CFU/ml. 300 .mu.l
and 500 .mu.l aliquots were dispensed into 3 ml pharmaceutical
vials for foaming and freeze-drying procedures respectively.
[0119] The bacteria were dried under vacuum without freezing using
the Q-T4 foaming protocol as described Example 2. The bacteria were
freeze-dried using the following protocol: ramp at 2.5.degree.
C./min to an initial shelf temperature of -40.degree. C.; primary
drying was performed at a vacuum pressure of 30 mT at -40.degree.
C., held for 40 hours; secondary drying was performed by a ramp at
0.05.degree. C./min from -40 to 30.degree. C. and holding for 12
hours.
[0120] Viability was determined immediately before and after the
completion of the drying procedures and after 3 weeks storage at
37.degree. C. using a plate assay as described in Example 2. The
residual moisture content and the glass transition temperature were
also determined.
[0121] The results for storage at 37.degree. C. are shown in Table
3. No significant loss in bacterial viability was observed after 3
weeks storage at 37.degree. C. in either the bacteria dried by the
Q-T4 method or the freeze-dried bacteria. The residual moisture
content and the glass transition temperature for the Q-T4-dried
bacteria was 1.85.+-.0.2% and 69.05.+-.5.0.degree. C. respectively.
The Tg for the freeze-dried bacteria was 104.5.+-.2.1.degree. C.
and the residual moisture content was 0.70.+-.0.2%.
3TABLE 3 Comparison between Q-T4 drying and freeze drying on viable
cell recovery of E. coli 9484 after storage at 37.degree. C. %
Viable cell recovery after storage at 37.degree. C. Day 0 Week 3
Q-T4sys (Drying) 43.7 .+-. 10.3 45.1 .+-. 8.5 Q-T4sys (Control)
2.51 .+-. 0.2 <0.01 Freeze Drying 30.6 .+-. 3.6 30.1 .+-. 3.7
Freeze Drying (Control) 1.83 .+-. 0.6 <0.01
EXAMPLE 9
Intracellular Accumulation of Trehalose During Growth of S.
typhimurium at 37.degree. C. in a High Salt Medium
[0122] S. typhimurium 1344 was grown in batch culture in either
minimal Salmonella growth medium with or without 0.5M NaCl (NaCl,
29.22 g 1.sup.-1; (NH.sub.4).sub.2SO.sub.4, 0.66 g 1.sup.-1;
K.sub.2HPO.sub.4, 10.5 g 1.sup.-1; KH.sub.2PO.sub.4, 4.5g 1.sup.-1;
MgSO.sub.4, 0.1 g 1.sup.-1; tryptophan, 20 mg 1.sup.-1; glucose at
a final concentration of 2.5% w/v) for a period of 106 hours at
37.degree. C. Samples were removed periodically for protein
measurement by the Bradford Assay and intracellular trehalose
determination by HPLC as described in Example 7. Trehalose
concentrations were expressed in .mu.mol of trehalose (mg
protein).sup.-1.
[0123] Significant concentrations of trehalose were observed
between 30 and 76 hours after inoculation reaching a maximum of
0.53 .mu.mol of trehalose (mg protein).sup.-1 after 48 hours as
shown in FIG. 9.
EXAMPLE 10
Stabilization of S. typhimurium 1344 at 37.degree. C. Using
Trehalose
[0124] S. typhimurium 1344 was grown in batch culture in minimal
Salmonella growth medium with 0.5M NaCl. Cells were grown for 60
hours at 37.degree. C. in a shaking incubator and harvested by
centrifugation at early stationary phase. A control culture where
the basal medium contained no salt was also prepared and harvested
at stationary phase in which trehalose synthesis would be markedly
reduced, since there is no osmotic stress.
[0125] Two 25 ml aliquots from each flask were harvested by
centrifugation at 10,000 rpm for 10 minutes. Cell pellets were
resuspended and washed in the appropriate growth medium. The
resulting cell pellet was resuspended in 8 ml of 45% trehalose,
0.1% CMC (Blanose 7HF; Aqualon). The suspensions were then pooled
to a total volume of 16 ml with a typical cell density of
2-4.times.10.sup.9 CFU/ml and 300 .mu.l aliquots dispensed into 3
ml pharmaceutical vials. Bacteria were dried under vacuum without
freezing as described in Example 2. Foaming occurred between 60-120
minutes of initial drying.
[0126] Samples of cultures (3.times.1 ml) were removed for
trehalose determination and protein estimation by the Bradford
assay (3.times.10 ml; Bio-Rad). Trehalose concentration was
expressed as .mu.mol (mg protein).sup.-1 as described in Example 7.
Viability was determined immediately before and after the
completion of the drying procedure and at various times during
storage at 37.degree. C. using a plate assay as described in
Example 2. The residual moisture content and the glass transition
temperature were also determined.
[0127] The storage results at 37.degree. C. are shown in FIG. 10.
No significant loss in viability was observed after 6 weeks storage
in S. typhimurium 1344, which was osmotically induced to accumulate
intracellular trehalose. Significantly, greater than 99% loss was
observed for the non-induced bacteria.
EXAMPLE 11
Confirmation of the Presence of the Trehalose-6-phosphate Synthase
(otsA) Gene in E. coli (NCIMB 9484) and Salmonella spp
[0128] Extracted genomic DNA from E. coli 9484, S. typhimurium 1344
and S. typhi Ty21a were qualified by OD260/280 nm and agarose gel
analysis. Each DNA preparation was prepared separately to ensure no
cross-contamination. The DNA was then used to prepare PCR reactions
with degenerate primers (where every third base has been
substituted either with a selection of bases or an inosine to allow
for any sequence changes), Guessmer primers (sequence selection
based upon Salmonella specific codon usage) and E. coli primers
(based purely on E. coli sequence) as shown in Table 4. Each set
produced at least one positive reaction. The relevant fragments
were run on low melting point gels and purified.
4TABLE 4 otsA gene probes for E. coli, S. typhimurium 1344, and S.
typhi Ty21a otsA gene (fragment size/ Target DNA application)
Primer set used E. coli 9484 700 bp/Sequence E. coli based E. coli
9484 150 bp/Southern Probe E. coli based S. typhimurium 1344 700
bp/Sequence Guessmer (Salmonella codon usage) S. typhimurium 1344
150 bp/Southern probe Guessmer (Salmonella codon usage) S. typhi
Ty21a 700 bp/Sequence Guessmer (Salmonella codon usage) S. typhi
Ty21a 400 bp/Sequence E. coli based
[0129] The 700 bp fragments were ligated into pCR3.1 and then
transferred into component cells and sequenced. The resulting
sequence data for otsA showed a sequence homology of 77% between S.
typhimurium 1344 and E. coli 9484. The sequence data also
demonstrated that only 6 bases from a total of 715 were different
between the two Salmonella spp strains.
[0130] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
Sequence CWU 1
1
6 1 488 PRT Kluyveromyces lactis 1 Met Val Asn Gln Asp Ile Ser Lys
Leu Ser Leu Asn Glu Cys Pro Gly 1 5 10 15 Ser Val Ile Val Ile Ser
Asn Arg Leu Pro Val Thr Ile Lys Lys Asp 20 25 30 Glu Lys Thr Gly
Glu Tyr Glu Tyr Ser Met Ser Ser Gly Gly Leu Val 35 40 45 Thr Ala
Leu Gln Gly Leu Lys Lys Ser Thr Thr Phe Gln Trp Tyr Gly 50 55 60
Trp Pro Gly Leu Glu Val Pro Asp Glu Asp Lys Ala Lys Val Lys Arg 65
70 75 80 Glu Leu Leu Glu Lys Phe Asn Ala Ile Pro Ile Phe Leu Ser
Asp Glu 85 90 95 Val Ala Asp Leu His Tyr Asn Gly Phe Ser Asn Ser
Ile Leu Trp Pro 100 105 110 Leu Phe His Tyr His Pro Gly Glu Ile Thr
Phe Asp Asp Thr Ala Trp 115 120 125 Leu Ala Tyr Asn Glu Ala Asn Met
Ala Phe Ala Asp Glu Ile Glu Gly 130 135 140 Asn Ile Asn Asp Asn Asp
Val Val Trp Val His Asp Tyr His Leu Met 145 150 155 160 Leu Leu Pro
Glu Met Ile Arg Gln Arg Val Ile Ala Lys Lys Leu Lys 165 170 175 Asn
Ile Lys Ile Gly Trp Phe Leu His Thr Pro Phe Pro Ser Ser Glu 180 185
190 Ile Tyr Arg Ile Leu Pro Val Arg Gln Glu Ile Leu Lys Gly Val Leu
195 200 205 Ser Cys Asp Leu Ile Gly Phe His Thr Tyr Asp Tyr Ala Arg
His Phe 210 215 220 Leu Ser Ala Val Gln Arg Ile Leu Asn Val Asn Thr
Leu Pro Asn Gly 225 230 235 240 Val Glu Phe Asp Gly Arg Phe Val Asn
Val Gly Ala Phe Pro Ile Gly 245 250 255 Ile Asp Val Glu Thr Phe Thr
Glu Gly Leu Lys Gln Asp Ala Val Ile 260 265 270 Lys Arg Ile Lys Glu
Leu Lys Glu Ser Phe Lys Gly Cys Lys Ile Ile 275 280 285 Ile Gly Val
Asp Arg Leu Asp Tyr Ile Lys Gly Val Pro Gln Lys Leu 290 295 300 His
Ala Leu Glu Val Phe Leu Gly Ala His Pro Glu Trp Ile Gly Lys 305 310
315 320 Val Val Leu Val Gln Val Ala Val Pro Ser Arg Gly Asp Val Glu
Glu 325 330 335 Tyr Gln Tyr Leu Arg Ser Val Val Asn Glu Leu Val Gly
Arg Ile Asn 340 345 350 Gly Gln Phe Gly Thr Ala Glu Phe Val Pro Ile
His Phe Met His Arg 355 360 365 Ser Ile Pro Phe Gln Glu Leu Ile Ser
Leu Tyr Ala Val Ser Asp Val 370 375 380 Cys Leu Val Ser Ser Thr Arg
Asp Gly Met Asn Leu Val Ser Tyr Glu 385 390 395 400 Tyr Ile Ser Cys
Gln Glu Glu Lys Lys Gly Thr Leu Ile Leu Ser Glu 405 410 415 Phe Thr
Gly Ala Ala Gln Ser Leu Asn Gly Ala Leu Ile Val Asn Pro 420 425 430
Trp Asn Thr Asp Asp Leu Ala Glu Ser Ile Asn Glu Ala Leu Thr Val 435
440 445 Pro Glu Glu Lys Arg Ala Ala Asn Trp Glu Lys Leu Tyr Lys Tyr
Ile 450 455 460 Ser Lys Tyr Thr Ser Ala Phe Trp Gly Glu Asn Phe Val
His Glu Leu 465 470 475 480 Tyr Arg Leu Gly Ser Ser Asn Asn 485 2
495 PRT Saccharomyces cerevisiae 2 Met Thr Thr Asp Asn Ala Lys Ala
Gln Leu Thr Ser Ser Ser Gly Gly 1 5 10 15 Asn Ile Ile Val Val Ser
Asn Arg Leu Pro Val Thr Ile Thr Lys Asn 20 25 30 Ser Ser Thr Gly
Gln Tyr Glu Tyr Ala Met Ser Ser Gly Gly Leu Val 35 40 45 Thr Ala
Leu Glu Gly Leu Lys Lys Thr Tyr Thr Phe Lys Trp Phe Gly 50 55 60
Trp Pro Gly Leu Glu Ile Pro Asp Asp Glu Lys Asp Gln Val Arg Lys 65
70 75 80 Asp Leu Leu Glu Lys Phe Asn Ala Val Pro Ile Phe Leu Ser
Asp Glu 85 90 95 Ile Ala Asp Leu His Tyr Asn Gly Phe Ser Asn Ser
Ile Leu Trp Pro 100 105 110 Leu Phe His Tyr His Pro Gly Glu Ile Asn
Phe Asp Glu Asn Ala Trp 115 120 125 Leu Ala Tyr Asn Glu Ala Asn Gln
Thr Phe Thr Asn Glu Ile Ala Lys 130 135 140 Thr Met Asn His Asn Asp
Leu Ile Trp Val His Asp Tyr His Leu Met 145 150 155 160 Leu Val Pro
Glu Met Leu Arg Val Lys Ile His Glu Lys Gln Leu Gln 165 170 175 Asn
Val Lys Val Gly Trp Phe Leu His Thr Pro Phe Pro Ser Ser Glu 180 185
190 Ile Tyr Arg Ile Leu Pro Val Arg Gln Glu Ile Leu Lys Gly Val Leu
195 200 205 Ser Cys Asp Leu Val Gly Phe His Thr Tyr Asp Tyr Ala Arg
His Phe 210 215 220 Leu Ser Ser Val Gln Arg Val Leu Asn Val Asn Thr
Leu Pro Asn Gly 225 230 235 240 Val Glu Tyr Gln Gly Arg Phe Val Asn
Val Gly Ala Phe Pro Ile Gly 245 250 255 Ile Asp Val Asp Lys Phe Thr
Asp Gly Leu Lys Lys Glu Ser Val Gln 260 265 270 Lys Arg Ile Gln Gln
Leu Lys Glu Thr Phe Lys Gly Cys Lys Ile Ile 275 280 285 Val Gly Val
Asp Arg Leu Asp Tyr Ile Lys Gly Val Pro Gln Lys Leu 290 295 300 His
Ala Met Glu Val Phe Leu Asn Glu His Pro Glu Trp Arg Gly Lys 305 310
315 320 Val Val Leu Val Gln Val Ala Val Pro Ser Arg Gly Asp Val Glu
Glu 325 330 335 Tyr Gln Tyr Leu Arg Ser Val Val Asn Glu Leu Val Gly
Arg Ile Asn 340 345 350 Gly Gln Phe Gly Thr Val Glu Phe Val Pro Ile
His Phe Met His Lys 355 360 365 Ser Ile Pro Phe Glu Glu Leu Ile Ser
Leu Tyr Ala Val Ser Asp Val 370 375 380 Cys Leu Val Ser Ser Thr Arg
Asp Gly Met Asn Leu Val Ser Tyr Glu 385 390 395 400 Tyr Ile Ala Cys
Gln Glu Glu Lys Lys Gly Ser Leu Ile Leu Ser Glu 405 410 415 Phe Thr
Gly Ala Ala Gln Ser Leu Asn Gly Ala Ile Ile Val Asn Pro 420 425 430
Trp Asn Thr Asp Asp Leu Ser Asp Ala Ile Asn Glu Ala Leu Thr Leu 435
440 445 Pro Asp Val Lys Lys Glu Val Asn Trp Glu Lys Leu Tyr Lys Tyr
Ile 450 455 460 Ser Lys Tyr Thr Ser Ala Phe Trp Gly Glu Asn Phe Val
His Glu Leu 465 470 475 480 Tyr Ser Thr Ser Ser Ser Ser Thr Ser Ser
Ser Ala Thr Lys Asn 485 490 495 3 517 PRT Aspergillus niger 3 Met
Pro Ser Leu Glu Asn Pro Thr Phe Gln Asn Glu Ala Arg Leu Leu 1 5 10
15 Leu Val Ser Asn Arg Leu Pro Ile Thr Ile Lys Arg Ser Asp Asp Gly
20 25 30 Arg Tyr Asp Phe Ser Met Ser Ser Gly Gly Leu Val Ser Gly
Leu Ser 35 40 45 Gly Leu Ser Lys Ser Thr Thr Phe Gln Trp Tyr Gly
Trp Pro Gly Leu 50 55 60 Glu Val Pro Glu Glu Glu Ile Pro Val Val
Lys Glu Arg Leu Lys Gln 65 70 75 80 Glu Tyr Asn Ala Val Pro Val Phe
Ile Asp Asp Glu Leu Ala Asp Arg 85 90 95 His Tyr Asn Gly Phe Ser
Asn Ser Ile Leu Trp Pro Leu Phe His Tyr 100 105 110 His Pro Gly Glu
Ile Thr Phe Asp Glu Ser Ala Trp Glu Ala Tyr Lys 115 120 125 Glu Ala
Asn Arg Leu Phe Ala Lys Ala Val Ala Lys Glu Val Gln Asp 130 135 140
Gly Asp Leu Ile Trp Val His Asp Tyr His Leu Met Leu Leu Pro Glu 145
150 155 160 Met Leu Arg Glu Glu Ile Gly Asp Ser Lys Glu Asn Val Lys
Ile Gly 165 170 175 Phe Phe Leu His Thr Pro Phe Pro Ser Ser Glu Ile
Tyr Arg Ile Leu 180 185 190 Pro Val Arg Asn Glu Leu Leu Leu Gly Val
Leu His Cys Asp Leu Ile 195 200 205 Gly Phe His Thr Tyr Asp Tyr Thr
Arg His Phe Leu Ser Ala Cys Ser 210 215 220 Arg Leu Leu Gly Leu Thr
Thr Thr Pro Asn Gly Ile Glu Phe Gln Gly 225 230 235 240 Lys Ile Ile
Ala Cys Gly Ala Phe Pro Ile Gly Ile Asp Pro Glu Lys 245 250 255 Phe
Glu Glu Gly Leu Lys Lys Glu Lys Val Gln Lys Arg Ile Ala Met 260 265
270 Leu Glu Gln Lys Phe Gln Gly Val Lys Leu Met Val Gly Val Asp Arg
275 280 285 Leu Asp Tyr Ile Lys Gly Val Pro Gln Lys Leu His Ala Leu
Glu Val 290 295 300 Phe Leu Ser Asp His Pro Glu Trp Val Gly Lys Val
Val Leu Val Gln 305 310 315 320 Val Ala Val Pro Ser Arg Gln Asp Val
Glu Glu Tyr Gln Asn Leu Arg 325 330 335 Ala Val Val Asn Glu Leu Val
Gly Arg Ile Asn Gly Lys Phe Gly Thr 340 345 350 Val Glu Phe Met Pro
Ile His Phe Leu His Lys Ser Val Asn Phe Asp 355 360 365 Glu Leu Ile
Ala Leu Tyr Ala Val Ser Asp Ala Cys Ile Val Ser Ser 370 375 380 Thr
Arg Asp Gly Met Asn Leu Val Ala Tyr Glu Tyr Ile Ala Thr Gln 385 390
395 400 Lys Lys Arg His Gly Val Leu Val Leu Ser Glu Phe Ala Gly Ala
Ala 405 410 415 Gln Ser Leu Asn Gly Ser Ile Ile Ile Asn Pro Trp Asn
Thr Glu Glu 420 425 430 Leu Ala Gly Ala Tyr Gly Glu Ala Val Thr Met
Ser Asp Glu Gln Arg 435 440 445 Ala Leu Asn Phe Ser Lys Leu Asp Lys
Tyr Val Asn Lys Tyr Thr Ser 450 455 460 Ala Phe Trp Gly Gln Ser Phe
Val Thr Glu Leu Thr Arg Ile Ser Glu 465 470 475 480 His Ser Ala Glu
Lys Phe His Ala Lys Lys Ala Ser Phe Ser Asp Asn 485 490 495 Asn Ser
Glu Asn Gly Glu Pro Ser Asn Gly Val Glu Thr Pro Ala Gln 500 505 510
Glu Gln Val Ala Gln 515 4 479 PRT Schizosaccharomyces pombe 4 Met
Ser Asp Ala His Asp Thr Ile Lys Ser Leu Thr Gly Asp Ala Ser 1 5 10
15 Asn Ser Arg Arg Leu Ile Val Val Ser Asn Arg Leu Pro Ile Thr Ile
20 25 30 Lys Arg Lys Asp Asn Gly Thr Tyr Asp Phe Ser Met Ser Ser
Gly Gly 35 40 45 Leu Val Ser Ala Leu Ser Gly Leu Lys Lys Leu Met
Thr Phe Gln Trp 50 55 60 Leu Gly Trp Cys Gly Gln Glu Ile Pro Glu
Asp Glu Lys Pro Met Ile 65 70 75 80 Ile Gln Arg Leu Gln Asp Glu Cys
Ser Ala Ile Pro Val Phe Leu Asp 85 90 95 Asp Glu Thr Ala Asp Arg
His Tyr Asn Gly Phe Ser Asn Ser Ile Leu 100 105 110 Trp Pro Leu Phe
His Tyr His Pro Gly Glu Ile Asn Phe Asp Glu Glu 115 120 125 Asn Trp
Glu Ala Tyr Arg Ala Ala Asn Tyr Ala Phe Ala Glu Ala Ile 130 135 140
Val Lys Asn Leu Gln Asp Gly Asp Leu Ile Trp Val Gln Asp Val His 145
150 155 160 Leu Met Val Leu Pro Gln Met Leu Arg Glu Leu Ile Gly Asp
Lys Phe 165 170 175 Lys Asp Ile Lys Ile Gly Phe Phe Leu His Thr Pro
Phe Pro Ser Ser 180 185 190 Glu Ile Tyr Arg Val Leu Pro Val Arg Asn
Glu Ile Leu Glu Gly Val 195 200 205 Leu Asn Cys Asp Leu Val Gly Phe
His Thr Tyr Asp Tyr Ala Arg His 210 215 220 Phe Leu Ser Ala Cys Ser
Arg Ile Leu Asn Leu Ser Thr Leu Pro Asn 225 230 235 240 Gly Val Glu
Tyr Asn Gly Gln Met Val Ser Val Gly Thr Phe Pro Ile 245 250 255 Gly
Ile Asp Pro Glu Lys Phe Ser Asp Ala Leu Lys Ser Asp Val Val 260 265
270 Lys Asp Arg Ile Arg Ser Ile Glu Arg Arg Leu Gln Gly Val Lys Val
275 280 285 Ile Val Gly Val Asp Arg Leu Asp Tyr Ile Lys Gly Val Pro
Gln Lys 290 295 300 Phe His Ala Phe Glu Val Phe Leu Glu Gln Tyr Pro
Glu Trp Val Gly 305 310 315 320 Lys Val Val Leu Val Gln Val Ala Val
Pro Ser Arg Gln Asp Val Glu 325 330 335 Glu Tyr Gln Asn Leu Arg Ala
Val Val Asn Glu Leu Val Gly Arg Ile 340 345 350 Asn Gly Arg Phe Gly
Thr Val Glu Tyr Thr Pro Ile His Phe Leu His 355 360 365 Lys Ser Val
Arg Phe Glu Glu Leu Val Ala Leu Tyr Asn Val Ser Asp 370 375 380 Val
Cys Leu Ile Thr Ser Thr Arg Asp Gly Met Asn Leu Val Ser Tyr 385 390
395 400 Glu Tyr Ile Cys Thr Gln Gln Glu Arg His Gly Ala Leu Ile Leu
Ser 405 410 415 Glu Phe Ala Gly Ala Ala Gln Ser Leu Asn Gly Ser Ile
Val Ile Asn 420 425 430 Pro Trp Asn Thr Glu Glu Leu Ala Asn Ser Ile
His Asp Ala Leu Thr 435 440 445 Met Pro Glu Lys Gln Arg Glu Ala Asn
Glu Asn Lys Leu Phe Arg Tyr 450 455 460 Val Asn Lys Tyr Thr Ser Gln
Phe Trp Gly Pro Lys Leu Cys Arg 465 470 475 5 498 PRT Mycobacterium
leprae 5 Met Thr Ser Arg Gly Asn His Gly Ser Lys Thr Ser Ser Asp
Lys His 1 5 10 15 Leu Gly Asp Ser Asp Phe Val Val Val Ala Asn Arg
Leu Pro Val Asp 20 25 30 Gln Val Arg Leu Pro Asp Gly Thr Ala Ile
Trp Lys Arg Ser Pro Gly 35 40 45 Gly Leu Val Thr Ala Leu Glu Pro
Leu Leu Arg Gln Arg Arg Gly Ala 50 55 60 Trp Val Gly Trp Pro Gly
Val Ile Asn Asp Asn Val Asp Leu Asp Leu 65 70 75 80 Thr Ile Lys Ser
Ile Val Gln Asp Gly Leu Thr Leu Tyr Pro Val Arg 85 90 95 Leu Asn
Thr His Asp Val Ala Glu Tyr Tyr Glu Gly Phe Ser Asn Ala 100 105 110
Thr Leu Trp Pro Leu Tyr His Asp Val Ile Val Lys Pro Ile Tyr His 115
120 125 Cys Glu Trp Trp Glu Arg Tyr Val Asp Val Asn Arg Arg Phe Ala
Glu 130 135 140 Thr Thr Ser Arg Thr Ala Ala Tyr Gly Gly Thr Val Trp
Val Gln Asp 145 150 155 160 Tyr Gln Leu Gln Leu Val Pro Lys Met Leu
Arg Ile Met Arg Pro Asp 165 170 175 Leu Thr Ile Gly Phe Phe Leu His
Ile Pro Phe Pro Pro Val Glu Leu 180 185 190 Phe Met Gln Ile Pro Trp
Arg Thr Glu Ile Ile Glu Gly Leu Leu Gly 195 200 205 Ala Asp Leu Val
Gly Phe His Leu Thr Ser Gly Ala Gln Asn Phe Leu 210 215 220 Phe Leu
Ser Arg His Leu Leu Gly Ala Asn Thr Ser Arg Gly Leu Val 225 230 235
240 Gly Val Arg Ser Arg Phe Gly Glu Val Gln Leu Lys Ser His Thr Val
245 250 255 Gln Val Gly Ala Phe Pro Ile Ser Ile Asp Ser Lys Glu Ile
Asp Gln 260 265 270 Ala Thr Arg Asp Arg Asn Val Arg Arg Arg Ala Arg
Glu Ile Arg Ala 275 280 285 Glu Leu Gly Asn Pro Arg Lys Ile Leu Leu
Gly Val Asp Arg Leu Asp 290 295 300 Tyr Thr Lys Gly Ile Asp Val Arg
Leu Arg Ala Phe Ala Glu Leu Leu 305 310 315 320 Ala Glu Gly Arg Ala
Lys Arg Asp Asp Thr Val Leu Val Gln Leu Ala 325 330 335 Thr Pro Ser
Arg Glu Arg Val Glu Ser Tyr Lys Ile Leu Arg Asn Asp 340 345 350 Ile
Glu Arg Gln Val Gly His Ile Asn Gly Glu Tyr Gly Glu Val Gly 355 360
365 His Pro Val Val His Tyr Leu His Arg Pro Ile Pro Arg Asp Glu Leu
370 375 380 Ile Ala Phe Tyr Val Ala Ser Asp Val Met Leu Val Thr Pro
Leu Arg 385 390 395 400 Asp Gly Met Asn Leu Val Ala Lys Glu Tyr Val
Ala Cys Arg Asn Asp 405 410 415 Leu Gly Gly Ala Leu Val Leu Ser Glu
Phe Thr Gly Ala Ala Ala Glu 420 425 430 Leu Arg Gln Ala Tyr Leu Val
Asn Pro His Asp Leu Glu Gly Val Lys 435 440 445 Asp Thr Ile Glu Ala
Ala Leu
Asn Gln Leu Ala Glu Glu Ala Arg Arg 450 455 460 Arg Met Arg Ser Leu
Arg Arg Gln Val Leu Ala His Asp Val Asp Arg 465 470 475 480 Trp Ala
Arg Ser Phe Leu Asp Ala Leu Ala Glu Ala Pro Ala Arg Asp 485 490 495
Ala Thr 6 473 PRT Escherischia coli 6 Ser Arg Leu Val Val Val Ser
Asn Arg Ile Ala Pro Pro Asp Glu His 1 5 10 15 Ala Ala Ser Ala Gly
Gly Leu Ala Val Gly Ile Leu Gly Ala Leu Lys 20 25 30 Ala Ala Gly
Gly Leu Trp Phe Gly Trp Ser Gly Glu Thr Gly Asn Glu 35 40 45 Asp
Gln Pro Leu Lys Lys Val Lys Lys Gly Asn Ile Thr Trp Ala Ser 50 55
60 Phe Asn Leu Ser Glu Gln Asp Leu Asp Glu Tyr Tyr Asn Gln Phe Ser
65 70 75 80 Asn Ala Val Leu Trp Pro Ala Phe His Tyr Arg Leu Asp Leu
Val Gln 85 90 95 Phe Gln Arg Pro Ala Trp Asp Gly Tyr Leu Arg Val
Asn Ala Leu Leu 100 105 110 Ala Asp Lys Leu Leu Pro Leu Leu Gln Asp
Asp Asp Ile Ile Trp Ile 115 120 125 His Asp Tyr His Leu Leu Pro Phe
Ala His Glu Leu Arg Lys Arg Gly 130 135 140 Val Asn Asn Arg Ile Gly
Phe Phe Leu His Ile Pro Phe Pro Thr Pro 145 150 155 160 Glu Ile Phe
Asn Ala Leu Pro Thr Tyr Asp Thr Leu Leu Glu Gln Leu 165 170 175 Cys
Asp Tyr Asp Leu Leu Gly Phe Gln Thr Glu Asn Asp Arg Leu Ala 180 185
190 Phe Leu Asp Cys Leu Ser Asn Leu Thr Arg Val Thr Thr Arg Ser Ala
195 200 205 Lys Ser His Thr Ala Trp Gly Lys Ala Phe Arg Thr Glu Val
Tyr Pro 210 215 220 Ile Gly Ile Glu Pro Lys Glu Ile Ala Lys Gln Ala
Ala Gly Pro Leu 225 230 235 240 Pro Pro Lys Leu Ala Gln Leu Lys Ala
Glu Leu Lys Asn Val Gln Asn 245 250 255 Ile Phe Ser Val Glu Arg Leu
Asp Tyr Ser Lys Gly Leu Pro Glu Arg 260 265 270 Phe Leu Ala Tyr Glu
Ala Leu Leu Glu Lys Tyr Pro Gln His His Gly 275 280 285 Lys Ile Arg
Tyr Thr Gln Ile Ala Pro Thr Ser Arg Gly Asp Val Gln 290 295 300 Ala
Tyr Gln Asp Ile Arg His Gln Leu Glu Asn Glu Ala Gly Arg Ile 305 310
315 320 Asn Gly Lys Tyr Gly Gln Leu Gly Trp Thr Pro Leu Tyr Tyr Leu
Asn 325 330 335 Gln His Phe Asp Arg Lys Leu Leu Met Lys Ile Phe Arg
Tyr Ser Asp 340 345 350 Val Gly Leu Val Thr Pro Leu Arg Asp Gly Met
Asn Leu Val Ala Lys 355 360 365 Glu Tyr Val Ala Ala Gln Asp Pro Ala
Asn Pro Gly Val Leu Val Leu 370 375 380 Ser Gln Phe Ala Gly Ala Ala
Asn Glu Leu Thr Ser Ala Leu Ile Val 385 390 395 400 Asn Pro Tyr Asp
Arg Asp Glu Val Ala Ala Ala Leu Asp Arg Ala Leu 405 410 415 Thr Met
Ser Leu Ala Glu Arg Ile Ser Arg His Ala Glu Met Leu Asp 420 425 430
Val Ile Val Lys Asn Asp Ile Asn His Trp Gln Glu Cys Phe Ile Ser 435
440 445 Asp Leu Lys Gln Ile Val Pro Arg Ser Ala Glu Ser Gln Gln Arg
Asp 450 455 460 Lys Val Ala Thr Phe Pro Lys Leu Ala 465 470
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