Field of the Invention
The invention is related to fermentation process for the production of heterologous
proteins in E coli expression system. The invention is also related to the process for
the production of heterologous proteins in E coli using pBAD24T7g10 vector. The
invention is further related to the vector pBAD24T7g10. The invention is also related to
the process of preparation of the vector pBAD24T7g10.
Background of the Invention
Production of heterologous proteins in E coli by recombinant DNA technology has
become the choice for expression systems to most of the researchers. It has certain
advantages over other systems, such as very well characterized, relatively simple
genetics, high growth, production rate and low cost etc. However, the disadvantages
are not less, like no post-translational modification and most importantly high level of
expression causes the formation of insoluble protein aggregates (known as inclusion
bodies) which are mostly inactive [Harrison: Innovations, 11:4-7 (2000)]. In order to get
an active protein, optimization of the expression conditions or the refolding studies are
required which could be time consuming and expensive. Various bacterial promoters
like Tac, T7, Trp, lacUV5, araBAD have been used for the heterologous protein
expression in E coli (Guzman et. al.1995 J. Bacteriol 177:4121-30, Cronan et. al.
2006,55:152-7).
Improving productivity is the major objective of fermentation in research and industry.
Productivity is a function of cell density and specific cell productivity. Specific cell
productivity refers to the amount of product formed per unit cell mass per unit time.
Thus increasing the cell density as well as specific cell productivity increases overall
fermentation productivity. Development of high cell density fermentation processes has
resulted in increased volumetric productivity of recombinant products in E coli. Yee
and Blanch, Biotechnology and Bioengineering, 41: 221-230 (1993). High cell density
cultures offer an efficient means for the economical production of recombinant
proteins. Depending upon the mode of operation and host cell system being employed,
a defined balanced batch and/or feed medium must be devised which will allow for cell
growth and expression of the recombinant protein (Khalilzadeh et al Iran. J.
Biotechnol. 2008 6(2), 63-84). The widely used inducer in high cell density cultivation
is isopropyl- -D-thiogalactopyranoside (IPTG), which is expensive for the large scale
production.
Use of arabinose induced pBAD promoter in the high cell density cultivation has been
described by Khalilzadeh et al (Iran. J. Biotechnol. (2008) 6(2), 63-84) as having the
disadvantage of product quality decreasing with cell density thus making it a bad
choice for the high cell density fermentations. pBAD24 expression vector is reported
by Guzman in 1995 (Guzman et al., J. Bacteriol. 177, 41214230, 1995; EMBL
accession number X81838). The pBAD24 vector has been reported to produce
proteins by researchers only at the lab scale, there is no report of pBAD24 being used
in a high cell density fermentation preparation of protein. Also it has been reported that
pBAD24 gives low yields of proteins for example aminopeptidase N, Staphylococcus
aureus lysylphosphatidylglycerol synthetase, glutamate transport protein and putative
O-antigen flippase (Golich 2006, Oku 2004, Jin 2006, Nelson 2006, Marolda 2004).
The present inventors while working with the pBAD24 expression vector, did not
observe any expression of proteins in spite of using the optimal inducer concentration
as reported by Guzman et. al 1995.
Fed-batch fermentation for the production of G-CSF in E.coli has been reported in
several patent documents for example in PCT publications WO 2006/067793A1; WO
2004/001056A1; WO 2007/102174A2; WO 2008/096368A2; and US patent application
US 2007/0015248A1.
The inventors while continuing their work with the pBAD24 vectors noticed that when
the pBAD24 vector is modified as described herein results in good yields of protein of
interest.
Summary of the Invention
In one aspect the invention is related to an industrial process for the production of
protein of interest from E. coli expression system comprising pBAD24T7g10 vector.
In another aspect the invention is related to an industrial process for the production of
protein of interest in K12 cells of E coli expression system comprising pBAD24T7g10
vector.
In another aspect the invention is related to the modified pBAD24 vector called as
pBAD24T7g10 having the following sequence in the 5' UTR of the translational start
site shown in SEQ ID 1.
5' GCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GGT ATA CAT ATG 3'.
In yet another aspect the invention is related to the construction of the pBAD24T7g10
vector.
In another aspect the invention is related to the fermentation process for the
production of protein of interest from E. coli, the process comprises the steps:
a) inoculating the fermenter with E. coli culture comprising pBAD24T7g10 vector
encoding protein of interest;
b) adding suitable production medium in the substrate limiting fed batch mode;
c) inducing the fermentation with a suitable inducer;
c) adding feed solution; and
e) subjecting the cells for cell lysis to obtain inclusion bodies comprising the protein
of interest.
In an aspect the fermentation is induced with a suitable inducer after about 4 to 5 hrs
of inoculation or when the OD600 reaches ~15.
In an aspect the feed solution is added to the fermentation medium after 7 to 8 hrs
from inoculation.
In an aspect the process of the invention involves addition of the feed solution at such
a rate that the OD600 of fermentation solution is increased to more than 100 ± 5 over a
period of about 10 to about 13 hours.
In one aspect the invention is further related to the production of pure protein of
interest from the inclusion bodies obtained from the cell lysis after fermentation step.
In another aspect the invention also relates to pharmaceutical composition comprising
therapeutically effective amount of the biologically active protein of interest obtained
according to the process of the present invention.
The details of one or more embodiments of the inventions are set forth in the
description below. Other features, objects and advantages of the inventions will be
apparent from the description and claims.
Brief Description of the Drawings
Fig. 1: Plasmid map of pBAD24T7g10
Fig. 2: Relationship between biomass increase and feed rate
Fig. 3: Expression of rhGCSF as an inclusion body
Detailed Description of the Invention
As used herein, "heterologous protein" or "protein of interest" refers generally to
peptides and proteins exogenous i.e. foreign to the E coli cells. Examples of the
protein includes molecules such as, colony stimulating factors (CSFs), for example M-
CSF, GM-CSF, and G-CSF; growth hormone, including human growth hormone;
interferon such as interferon-alpha, -beta, and -gamma; interleukins (ILs), such as IL-2,
IL-11, IL-1RA; reteplase, staphylokinase, Steptokinase DPP-4, DPP-8, PTH, PDGFAA,
PDGFAB, PDGFBB and fragments of any of the above-listed polypeptides.
As used herein, the term "inclusion bodies" refers to dense intracellular masses of
aggregated polypeptide of interest, which constitute a significant portion of the total cell
protein, including the cellular components. These aggregated polypeptides may be
incorrectly folded or partially correctly folded proteins.
The term "therapeutically effective amount" used herein refers to the amount of
biologically active protein which has the therapeutic effect of biologically active protein.
The term "biologically active protein" used herein refers to protein which is capable of
promoting the differentiation and proliferation of hematopoietic precursor cells and the
activation of mature cells of the hematopoietic system.
In an embodiment the invention provides a process for industrial preparation of
recombinant protein of interests from E. co//using pBAD24T7g 10 vector.
According to one embodiment of the invention there is provided a high cell density
fermentation process for the production of protein of interests from E coli using
pBAD24T7g 10 vector.
The proteins synthesized using the process of the invention includes but not limited to
GCSF, reteplase, interleukins, human growth hormone, DPP-4, DPP-8,
staphylokinase, interferon, teriparatide.
Construction of the expression vector pBAD24T7g10:
The pBAD24 vector is digested with Nhel/EcoRI and ligated with the annealed oligo
carrying T7g10 sequence elements which are already been digested with the same set
of enzymes. This construction allows to retain all the MCS present in pBAD24 along
with translational enhancer sequence of T7 phage. To this vector the protein of
interest is cloned as EcoRI/Hindlll fragment with an internally created Ndel site which
provides initiation codon for translation. The Figure 1 shows the plasmid map of
pBAD24T7g10.
Though pBAD24T7g10 contains an inherent ampicillin (antibiotic) marker for selection
of transformants, other antibiotic markers like chloramphenicol (coded by
chloramphenicol acetyl trasnferase- CAT), Kanamycin (APH, phosphotransferases),
tetracycline resistance gene etc. can be used as antibiotic markers. These markers
can be cloned either under a constitutive promoter or under their own respective
promoters at suitable restriction sites of the pBAD24T7g10 vector.
Fermentation
E coli cells are transformed with suitable vector containing gene to synthesize rh-
protein. Various strains of E co//may be used for the process of the present invention
for example cells which are protease deficient strains such as BL21, ER2566 and the
protease expressing strains of K12 derivatives such as HB101, JM109, LE392, C600,
TOP10, DH5 alpha and the like. The culture of E.coli is used for the fermentation.
High expression of the proteins using the pBAD24T7g10 vector of the present
invention depends on various parameters of the fermentation process. Some of the
parameters are fermentation media, concentration of the inducer, nutrient feed rate.
Culture medium is prepared for different stages of the fermentation as described
below:
Preferably the feed medium comprises 1 to 15% carbon source and 1 to 20% nitrogen
source. The carbon source may comprise glucose, glycerol, sorbitol, maltose, sucrose
or starch, mannitol. Preferably the carbon source is mannitol, glucose or glycerol or
mixture thereof. The nitrogen source may comprise ammonia, nitrate, peptone, soya
peptone, yeast extract or tryptone. Preferably the nitrogen source is yeast extract or
soya peptone or tryptone or mixtures thereof.
Preferably the feed medium comprises of 0.005 to 0.02 % antibiotics and 1 to 10 % of
inorganic phosphates and trace elements.
The feed medium may comprise antibiotics such as ampicillin or tetracycline or any
other antibiotic such as kanamycin, tetracycline, chloramphenicol, hygromycin and the
like depending on the antibiotic marker of the vector.
Preferably, the expression of the protein of interests is induced at a cell density when
OD600 of the culture is between 10 to 20. Preferably the production is induced with the
L-(+)-arabinose as inducer.
The percentage of dissolved oxygen is adjusted between 20%-60%. Preferably the
temperature of the fermentation broth is maintained between 30 ° C to 44 ° C and
preferably at 37 °C. Preferably pH of the fermentation broth is maintained at pH 6.5 -
pH 7.5 and preferably at pH 7.0. The fermentation may be carried out for a period of
12 to 24 hours.
Preparation of seed culture:
Typically the seed medium is inoculated with glycerol stock of the culture. Inoculated
flask is incubated at 37 °C, 200 rpm for 16 to 18 h. Seed medium used herein is
composed of 1% Tryptone, 0.5% yeast extract, 1% sodium chloride suspended in
water and pH of the seed medium is in the range of 7.2 to 7.4
Preparation and inoculation of fermenter medium:
To a suitable fermenter is added the fermentation medium. The fermentation medium
is the medium required for the growth and expression of rh-proteins at fermenter scale.
Typically the fermentation medium comprises of suitable salts, carbon source and
nitrogen source while antifoam (at 0.05%) may also be added. The suitable salts
include ammonium chloride, potassium di-hydrogen phosphate, di-sodium hydrogen
phosphate, sodium chloride, magnesium sulphate and the like. Suitable carbon
sources include but not limited to one or more of mannitol, glucose, arabinose or
glycerol. Nitrogen sources which may be used include but not limited to one or more of
are yeast extract, tryptone, soya peptone and the like.
In an embodiment of the invention the fermentation is carried out in the presence of
zinc ions. During fermentation the presence of zinc ions help prevent bacteriophage
contaminations. Additionally the presence of zinc may help in the activation of the
methionine aminopeptidase activity in E.coli'as described in BBRC, 2003, 307, 172-79;
Protein Science, 1998, 7, 2684-87; both the references are incorporated herein in their
entirety.
In the present process of high cell density fermentation for production of recombinant
therapeutic proteins, non toxic surfactants are added such as Tween 20, Tween-80.
Tween-20 at concentration of 0.05% is added in the medium, considering its role to
prevent bacteriophage contamination in bacterial fermentations. The book,
Biotechnological Innovations in chemical synthesis by R. C. Van Dam Mieras, Chapter
8 Page No: 248 & a paper by D. Perlman, Wayne W. Umbreit Bacteriophages of the
genus Clostridium in a journal, Advances in Applied Microbiology, discloses addition of
non toxic surfactants to prevent bacteriophage contamination during bacterial
fermentations, the reference is incorporated herein.
Typically, the fermenter medium is prepared using 1 to 3 % tryptone, 1.5 to 6 % yeast
extract, 1 to 5 % mannitol, 0.05 to 0.2 % ammonium chloride, 0.1 to 0.5 % potassium
di-hydrogen phosphate, 0.5 to 3 % di-sodium hydrogen phosphate, 0.01 to 0.1 %
sodium chloride, 0.01 to 0.1 % magnesium sulphate, 0.00025% zinc, 0.01 to 0.2 %
Tween-20.
In an embodiment of the invention the suitable salts are dissolved in water in a transfer
flask. The required quantities of yeast extract and tryptone are mixed in water and
added to a fermenter vessel. Similarly required quantity of mannitol is dissolved in
water in a transfer flask. Magnesium sulphate is dissolved in another transfer flask. All
the transfer flasks and the fermenter are sterilized at 121 °C for 30 minutes in an
autoclave. Fermenter media is reconstituted aseptically after all the solutions in
transfer flasks are cooled.
Feed solution used for growth and expression of rh-proteins at fermenter scale is
prepared as follows. The feed solution comprises of suitable salts, carbon source,
nitrogen source, inducer and antibiotic.
Typically the concentration of the feed solution comprises of 1 to 5 % Tryptone, 5 to 20
% Yeast Extract, 5 to 20 % Mannitol, 0.1 to 0.5 % Ammonium chloride, 0.5 to 2 %
Potassium di-hydrogen phosphate, 2 to 8 % di-Sodium hydrogen phosphate, 0.025 to
0.3 % Sodium chloride, 0.025 to 0.3 % Magnesium sulphate. L-(+)-arabinose is added
as inducer. Inducer is used in the range of about 3 to 25 mM.
The feed solution is prepared by dissolving required quantities of salts in water as
solution A. The required quantities of yeast extract and tryptone are mixed and
dissolved in water as solution B. The solution of carbon source, mannitol is prepared
by dissolving in water as solution C. Solution D is prepared by dissolving magnesium
sulphate in water. All the solutions A, B, C and D in separate flasks are sterilized
separately at 121 °C in an autoclave and mixed together. To the prepared feed
solution L-(+)-arabinose and antibiotic such as ampicillin is also added.
The fermenter used for the invention is microbial fermenter having provision for oxygen
enrichment and is equipped with control devices comprising of sensors & controllers
for temperature, pH and oxygen while pumps for addition of feed medium, acid/base,
inducer and antifoam solutions. The pH is maintained using buffers preferably 30%
Phosphoric acid or 12% Ammonia solution. Antifoam used is 1510-US from Dow
corning & is added drop wise as per requirement.
Fermenter with sterilized media is connected to its control panel; pH and DO probes
and feed pumps are checked for their calibration. Initially agitation is set at 300 rpm
and is increased to maximum of 800 rpm as to maintain dissolved oxygen in a range of
20 to 60 %. Aeration is maintained at 1vvm of air. If required, air is supplemented with
oxygen gas in the proportion set by automatic controller of the instrument so as to
maintain dissolved oxygen in a range of 20 to 60 %. Temperature is maintained at
37°C by PID controller of the instrument. Acid, base and sterile antifoam solution
bottles are connected to respective ports on to a fermenter with the help of silicon
tubing and pumps. pH is maintained at 7.00 by automatic addition of acid or base with
the help of PID controller of the instrument.
Before inoculation of seed culture in a fermenter media, Filter sterilized ampicillin
solution is added at concentration of 100 ng/ml.
To start the fermentation process, 5 to 20 % V/v seed culture is inoculated to 2-liter
fermenter medium. Batch is monitored for its pH, optical cell density OD600 and other
process parameters like temperature, % of dissolved oxygen, agitation, volume of acid,
base or feed consumed, etc at the interval of every one hour. After about 4 to 5 hours
of inoculation, when OD600 of the culture is between 10 to 20, it is induced with L-(+)-
arabinose. A first dose of 10 ml stock solution (10%) of L-(+)-arabinose is added to
growing culture, followed by same dose at an interval of 1 hour up to 6 hours.
Three hours after induction of culture process is shifted as a fed batch process.
Volumetric addition of feed solution is as given in a table below.

The feed solution is added at such a rate that the OD600 of fermentation solution is
increases to more than 100 ± 5 over a period of about 10 to about 13 hours;
Cell pellet of 2 liter fermented broth is suspend in 800 ml to 1 liter of 10 mM Tris pH 8.0
and is mixed so as to get uniform mixture, having initial OD600 between 100 to 150.
Suspended cell pellet is subjected to homogenization in a homogenizer.
Homogenization is carried out at a pressure of 800 to 900 bars and is done for 3 to 4
cycles, which is for about 20 minutes. The entire operation is done under cooling
conditions. The fall in optical density of cell suspension is between 80 to 85%.
After this the cell lysate is chilled to about 5 to 8 °C on ice for 30 mins & it is subjected
to centrifugation at 9000 RPM for 30 minutes at 4 °C. Supernatant is discarded while
pellet i.e. inclusion bodies of proteins are preserved at -20 °C until further use for
purification.
In an embodiment the invention further provides a process for production of pure
protein of interest from the inclusion bodies. For obtaining the pure proteins from the
inclusion bodies one skilled in the art can follow the procedures described in the
literature. Typically the process for the production of pure protein of interest from the
inclusion bodies includes solubilizing the inclusion bodies of proteins; refolding the said
solubilized proteins; purifying the refolded proteins; and isolating pure proteins. There
are various methods reported in the literature, one skilled in the art can follow one or
more methods to obtain protein of interest.
Purification methods include but not limited to aqueous two phase extraction, various
chromatography techniques. The purification of GCSF is disclosed in our copending
patent application No. IN 865/KOL/2009 filed on June 16, 2009, which is incorporated
herein by reference in its entirety). The protein obtained using the aqueous two phase
extraction process may be further processed, to provide the protein or polypeptide
having high purity. Further purification may be necessary to remove related impurities.
The impurities may include oxidized forms, deamidated forms, aggregated proteins
and also degraded forms such as biologically inactive monomeric forms, incorrectly
folded protein molecules, denaturated forms of protein, host cell proteins, host cell
substances such as DNAs, (lipo)polysaccharides etc and additives which had been
used in the preparation and processing of proteins. Such higher purity may be required
depending on the use for which the protein or polypeptide is intended. For example,
therapeutic uses of the protein will typically require further purification following the
extraction methods of the invention. All protein purification methods known to the
skilled artisan may be used for further purification. Such techniques have been
extensively described in Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Volume 152, Academic Press, San Diego, Calif. (1987);
Molecular Cloning: A Laboratory Manual, 2d ed., Sambrook, J., Fritsch, E. F., and
Maniatis, T. (1989); Current Protocols in Molecular Biology, John Wiley & Sons, all
Viols., 1989, and periodic updates thereof); New Protein Techniques: Methods in
Molecular Biology, Walker, J. M., ed., Humana Press, Clifton, N.J., 1988; and Protein
Purification: Principles and Practice, 3rd. Ed., Scopes, R. K., Springer-Verlag, New
York, N.Y., 1987, the above are incorporated herein by references in its entirety. In
general, techniques including, but not limited to, ammonium sulfate precipitation,
centrifugation, ion exchange, reverse-phase chromatography, affinity chromatography,
hydrophobic interaction chromatography may be used to further purify the protein.
The yield of the protein of interest from E coli using pBAD24T7g10 vector using the
processes of the invention are in the range of 20 to 35% of the total cellular protein in
the bacterial cell.
The process of obtaining pure protein of interest as described herein further comprises
of forming the pure proteins into a finished dosage form for clinical use.
The biologically active protein obtained by the entire process for the purification and/or
isolation of the present invention is suitable for the preparation of pharmaceutical
composition, which comprises the therapeutically effective amount of biologically
active protein and one or more pharmaceutical excipients and is suitable for clinical
use. The possibility of maintaining the active form of protein in a short purification and
isolation process contributes not only to an improved yield, but also to an improved
purity and effectiveness of the biologically active protein and the pharmaceutical
composition containing it. Suitable pharmaceutically acceptable excipients include but
not limited to suitable diluents, adjuvants and/or carriers and the similar useful in
protein therapy.
In yet another embodiment the invention relates to pharmaceutical compositions
containing the proteins obtained according to the present invention. The proteins
obtained can either be stored in the form of a lyophilisate or in liquid form. It is
administered either subcutaneously or intravenously.
The following examples are provided to further illustrate the present invention but are
not provided to in any way limit the scope of the current invention.
Example -1 : Construction of vector
pBAD24 was digested with Nhel/EcoRI and ligated with the annealed oligo carrying
T7g10 sequence elements that has already been digested with the same set of
enzymes. This construction would allow retaining all the MCS present in pBAD24
along with translational enhancer sequence of T7 phage. To this vector human GCSF
was cloned as EcoRI/Hindlll fragment with an internally created Ndel site that would
provide initiation codon for translation.
Culture used was genetically engineered Escherichia coli K 12 cells transformed with
suitable vector containing gene to synthesize rh-proteins.
Sequence listing of pBAD24T7g10-GCSF

Example -2: Fermentation
Different media used for growth and production (expression) of rh-GCSF were.
1) Seed medium was the medium used for initial growth of the culture from the glycerol
stock of it. Seed medium was composed of 1% Tryptone, 0.5% Yeast Extract 1%
Sodium chloride suspended in water while pH of this medium falls in the range of 7.2
to 7.4.
2) Fermenter medium used for growth and expression of rh-GCSF at fermenter scale,
it is composed of 1.8% tryptone, 3.6% yeast Extract, 1% Mannitol, 0.1% Ammonium
chloride, 0.3% Potassium di-hydrogen phosphate, 1.2% di-Sodium hydrogen
phosphate, 0.05% Sodium chloride, 0.05% Magnesium sulphate, 0.00025% zinc and
0.05% Tween-20.
3) Feed solution used for growth and expression of rh-GCSF at fermenter scale and is
composed of 3.9 % Tryptone, 7.8 % Yeast Extract, 7.8 % mannitol, 0.325%
Ammonium chloride, 0.976% Potassium di-hydrogen phosphate, 4.162% di-Sodium
hydrogen phosphate, 0.162% Sodium chloride, 0.162% Magnesium sulphate. Inducer
used was 20mM L-(+)-arabinose. Fermenter used was Biostat B plus (M/s Sartorius)
microbial fermenter having provision for oxygen enrichment. For maintaining the pH
during process either 30% Phosphoric acid or 12% Ammonia solution was used.
Antifoam used was 1510-US from Dow corning & was added drop wise as per
requirement
Preparation of seed culture: 200ml seed medium, composition as described above
was inoculated with glycerol stock of the culture. Inoculated flask was incubated on
rotary shaker at 37°C, 200 rpm for 16 to 18 hours.
Preparation and inoculation of fermenter medium: 2-liter Fermentation medium in a
fermenter vessel with 5-liter capacity was prepared as follows.
A-Required quantities of salts as described in the fermenter medium were dissolved in
700 ml of water and were added to a transfer flask.
B- Required quantities of yeast extract, tryptone & Tween-20 were mixed and
dissolved in 850ml of water. This solution was prepared in a fermenter vessel.
C- Required quantity of mannitol was dissolved in 200ml of water. This solution was
prepared in a transfer flask.
D- Required quantity of magnesium sulphate was dissolved in 50ml of water
A, B, C & D were sterilized separately at 121°C for 30 minutes in an autoclave, after
cooling all three were mixed aseptically in a fermenter.
Preparation of feed solution:
E-Required quantities of salts as described in the feed solution were dissolved in
200ml of water
F- Required quantities of Yeast extract and Tryptone were mixed and dissolved in
280ml of water.
G- Required quantity of Mannitol was dissolved in 250 ml of water.
H- Required quantity of MgS04 was dissolved in 50 ml of water.
E, F, G & H were prepared in the separate flasks, sterilized separately at 121°C for 30
minutes in an autoclave and were pooled together to make 800ml feed solution. In
addition to all this, 2.4 g inducer L-(+)-arabinose was dissolved in 20ml of water, filter
sterilized & was added to feed solution.
Filter sterilized Ampicillin solution was added to a feed solution as well as to a
fermenter batch medium at concentration of 100 (ig/ml. L-(+)-arabinose required for 2
liter of fermentation media at concentration of 20mM was dissolved in 60ml of water
and was filter sterilized, in a sterile transfer flask. Fermenter with sterilized media was
connected to its control panel; pH and DO probes and feed pumps were checked for
their calibration. Initially agitation was set at 300 rpm and was increased to maximum
of 800 rpm as to maintain dissolved oxygen above 30%. Aeration was maintained at
1vvm of air. If required, air was supplemented with oxygen gas in the proportion set by
automatic controller of the instrument so as to maintain dissolved oxygen above 30%.
Temperature was maintained at 37°C by PID controller of the instrument. Acid, base
and sterile antifoam solution bottles were connected to respective ports on to a
fermenter with the help of silicon tubing and pumps. pH was maintained at 7.00 by
automatic addition of acid or base with the help of PID controller of the instrument.
To start the fermentation process, 200 ml seed culture was inoculated to 2-liter
fermenter medium. Batch was monitored for its pH, optical cell density OD600 and other
process parameters like temperature, % of dissolved oxygen, agitation, volume of acid,
base or feed consumed, etc at the interval of every one hour. After about 4 to 5 hours
of inoculation, when OD600 of the culture reaches -15, culture was induced with L-(+)-
arabinose. A first dose of 10ml stock solution of L-(+)-arabinose was added to growing
culture, followed by same dose at an interval of 1 hour for 6 hours
Three hours after induction of culture, volume feed added to fermenter was as follows.

OD600 of culture was gradually increased to 106 over a period 10 to 13 hours.
Cell pellet of 2 liter fermented broth was suspend in 800 ml to 1 liter of 10 mM Tris pH
8.0 and was mixed so as to get uniform mixture of it having initial OD600 between 100
to 150. Suspended cell pellet was subjected to homogenization.
Homogenization was carried out at a pressure of 800 to 900 bars and was done for 3
to 4 cycles, which was about 20 minutes. The entire operation was done under cooling
conditions. Optical cell density falls in a range of 20 to 30. Cell lysate was chilled to
about 5 to 8 °C on ice for 30 mins.
Chilled cell lysate was subjected to centrifugation at 9000 RPM for 30 minutes at 4 °C.
Supernatant is discarded while pellet i.e. inclusion bodies are preserved at -20°C.
While the present invention has been described in terms of its specific embodiments,
certain modifications and equivalents will be apparent to those skilled in the art and are
intended to be included within the scope of the present invention.
We Claim:
1. A vector pBAD24T7g10 having the following sequence in the 5'UTR of the
translational start site, shown in SEQ ID 1,
5' GCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GGT ATA CAT ATG 3'
2. A process for the preparation of recombinant protein from E coli using the
pBAD24T7g 10 vector.
3. The process of claim 2, wherein the process comprising the steps of:
a) inoculating the fermenter with E coli culture comprising pBAD24T7g10 vector
encoding protein of interest;
b) adding suitable production medium in the substrate limiting fed batch mode;
c) inducing the fermentation with an inducer;
c) adding feed solution; and
e) subjecting the cells for cell lysis to obtain inclusion bodies comprising the protein
of interest.
4. The process as claimed in claim 2 or 3, wherein the E coli host cells are selected
from the group consisting of BL21, ER2566, K12 derivatives such as HB101,
JM109, LE392, C600, TOP10 and DH5 alpha.
5. The process as claimed in claim 2 or 3, further comprises obtaining the pure protein
of interest from the inclusions bodies.
6. The process as claimed in claim 3, wherein the inducer is arabinose.
7. The process as claimed in claim 3, wherein the feed solution is added after about 7
to 8 hrs from inoculation.
8. The process as claimed in claim 3, wherein the fermentation is induced with an
inducer after about 4 to 5 hrs from inoculation or when the OD600 is about 15.
9. The process as claimed in claim 3, wherein the fermentation is carried out in the
presence of zinc ions.
10. The process as claimed in claims 2 to 9, wherein the protein of interest is GCSF.
A B S T R A C T
TITLE : FERMENTATION PROCESS FOR THE PREPARATION OF
RECOMBINANT HETEROLOGOUS PROTEINS
The invention is related to high cell density fermentation process for production of
heterologous proteins in E. coli expression system. The invention is also related to the
process for the production of heterologous proteins in E. coli using pBAD24T7g10
vector. The invention is also related to the vector pBAD24T7g10. The invention is also
related to the process of preparation of the vector pBAD24T7g10. The fermentation may
be performed using E. coli K12 cells.

The invention is related to high cell density fermentation process for production of
heterologous proteins in E. coli expression system. The invention is also related to the
process for the production of heterologous proteins in E. coli using pBAD24T7g10
vector. The invention is also related to the vector pBAD24T7g10. The invention is also
related to the process of preparation of the vector pBAD24T7g10. The fermentation may
be performed using E. coli K12 cells.