bioprocess optimization and production of the...
TRANSCRIPT
CHAPTER3
BIOPROCESS OPTIMIZATION AND
PRODUCTION OF THE RECOMBINANT
LETHAL FACTOR USING FED-BATCH
CULTURE TECHNIQUE
Introduction
Genetic engineering methods are in industrial use to produce many types of
recombinant proteins using fast growing microorganisms such as E. coli. For the
economical use of these recombinant microorganisms that form intracellular
products, it is important to utilize a fermentation process that results in a high
intracellular level of product and a high cell concentration in the fermentor. Fed
batch culture techniques have been routinely employed to obtain high cell density
cultures of such strains producing recombinant products. In this chapter attempts
have been made to scale up the expression of the recombinant lethal factor by
bioprocess optimizing the growth of cultures carrying the recombinant plasmid
pPG-LFl. Attention has been focussed on increasing the productivity through an
increase in host cell mass i.e., high density cultivations but at the same time
strategies have been adopted to minimize organic acid production which inhibits
the growth of the micro-organism. The media, a defined mixture of salts, trace
elements, vitamins etc. alongwith a specified carbon source have been used for
growth in 14 litre fermentor. Dissolved oxygen (DO) has been maintained above
20 % by automatic control of agitation. Efforts have also been made to identify a
suitable C-source and selection of proper specific growth rate(s) for cultivation
and induction in fed batch culture.
Experimental Methodology
Growth Curve of the recombinant culture at 28°C and half the concentration
of desired antibiotics
E. coli SG 13009(pREP4) cells carrying the construct pPG-LF1 were inoculated in
10 ml LB medium containing 100 1-1g of ampicillin per rnl and 25 llg of kanamycin
per ml from the glycerol stock and grown overnight at 37oc at 250 rpm. Next
day, 1 % of the overnight grown culture was inoculated in 1 litre LB medium
containing 50 llg of ampicillin per ml and 12.5 llg of kanamycin per ml. The
flasks were incubated at 28°C I 250 rpm. Cultures were also grown in the
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presence of I 00 Jlg of ampicillin per ml and 25 Jlg of kanamycin per ml at 28°C as
well as 37oc. Samples were collected after every hour and optical density was
determined at 600 nm. Growth curve was plotted to determine the effect of
temperature and half antibiotic concentration on the growth of the E. coli cultures
containing the recombinant plasmid pPG-LFI.
Fermentor
A 14L (Chemap AG) fermentor was used for the work. It was equipped with pH,
temperature and dissolved oxygen monitoring and control. The fermentor was
interfaced with a personal computer. The minimum and maximum permissible
working volumes were 6L and 10L respectively. The maximum permissible
aeration was 2 vvm and agitation upto 1000 rpm could be achieved. An in-house
developed software was used for data acquisition and proper operation of the
ferementor both in batch as well as fed-batch mode. The software had the
capability of designing all the operational parameters of the fed-batch culture
based upon user's requirements. It permitted implementation of multiple specific
growth rates which was required in cultivation of recombinant cultures.
Preculture Medium
All chemicals were procured from Qualigens Fine Chemicals, India, excepting
yeast extract & tryptone which were procured from Hi-Media Laboratories, India.
The malic acid was obtained from SD Fine Chemicals, India. Silicone antifoam-A
concentrate from Sigma, USA after dilution (1 0% V N) in silicone fluid was used
as antifoam agent.
The medium for pre-cultures had the following composition.
(a). Tryptone 10.0 g/1
(b). Yeast extract 5.0 g/1
(c). NaCl 5.0 g/1
pH 6.8
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The inoculum for fermentor was prepared in two stages, namely preculture-1 (PC
I) and preculture-II (PC-II). For preparation of PC-I, 2 x 10 ml sterile medium in
2 x 100 ml flask was inoculated with a loopful from a glycerol stock. The culture
was allowed to grow in a shaker (250 rpm, 37°C) for 14h. For making PC-11, 2 x
200 ml sterile medium in 2 x 1L flask was inoculated with 10 ml from a 14h old
PC-I. After 14h, PC-II was used as inoculum for fermentor@ 5% VN for 6L
medium in fermentor.
Fermentor Medium
A typical fermentation media composition for batch and fed-batch phases is given
below. The components were autoclaved in groups to prevent precipitation.
No. Component
(a) KzHP04
(b) Citric acid
(c) KHzP04
(d) NaCl
(e) Yeast Extract
(f) Tryptone
(g) Trace Metals
(h) MgS04.6H20
(i) Ampicillin
(j) Kanamycin
(k) Thiamine. HCl
Concentration
Batch Medium
5.0 g/1
1.7 g/1
4.0 g/1
5.0 g/1
5.0 g/1
10.0 g/1
*cf next table
0.2 g/1
50 mg/1
13 mg/1
2 mg/1
Concentration
Feed Medium
100 g/1
100 g/1
3 g/1
100 mg/1
25 mg/1
------------------------------------------------------------------------------------------------------------
• Composition of trace metals stock solutions is given below:
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. ---------------------------------------------------------------------------------------------------
Component Concentration Stock Solution In6LMedium
(mg/1) (mg/ml)
F e(III)-citrate 50 12.00 4.1 ml
MnClz.4HzO 8 15.00 0.6 ml
ZnC}z 4 8.40 0.5 ml
H3B03 2 3.30 0.6 ml
NazMo04.2HzO 2 2.67 0.8 ml
CoClz.2HzO 2 2.76 0.7 ml
CuClz.2HzO 1 1.50 0.7 ml
EDT A-N ar2Hz0 10 8.40 1.2 ml
Batch Medium:
Group-I
Components (a) to (g) were dissolved in distilled water in the fermentor and .0.5
ml antifoam agent was added. The fermentor was sterilized (121°C, 45 min) with
indirect steam and cooled to 28°C.
Group-II
Component (h) was dissolved separately in flask and autoclaved (121 °C, 30 min).
On cooling it was added to the fermentor asceptically using transfer bottles.
Group-III
Components (i) to (k) were added to sterile & cooled fermentor as filter sterilized
(0.2).! Saritorius filter)
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Feed Medium
Group-IV
Component (e) & (f) were dissolved in flasks separately and autoclaved & cooled
to room temperature.
Group-V
Component (h) was dissolved in a flask. After autoclaving for 20 min, the flask
was cooled to room temperature.
Group-VI
Component (i) & (j) were dissolved separately and filter sterilized.
Finally group (IV), (V) and (VI) were pooled in a 5L sterilized polycarbonate
bottle which served as a feed reservoir.
Fermentor Operation in batch mode
After sterilization of the fermentor, the pH of the fermentor media was set to 6.8
by the addition of 4N NaOH I 4N H3P04 and temperature to 28°C. The fermentor
was started in batch phase with a working volume of 6L. The fermentor was
inoculated with overnight grown PC II. The DO during the growth phase was
controlled with increasing agitation. After 6 hr., the cultures were induced with 1
mM IPTG. After 11 hrs., the run was suspended and the cells were harvested.
Selection of alternate Carbon - source
E. coli SG 13009 (pREP4) cells containing the recombinant plasmid pPG-LF1
were grown in 1 litre LB modified medium in presence of different carbon sources
such as glucose, lactose, maltose and DL- malic acid. Glucose (0.1 %) was added
as alternate C-source in the modified LB medium. Equivalent amount (carbon) of
lactose, maltose and malic acid were added separately in the modified LB
medium. The antibiotic concentration in the media was 50 J-lg I ml of ampicillin
and 12.5 J-lg I ml of kanamycin. Cultures were grown at 28°C and samples were
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collected after every hour. Cultures were induced with 1 mM IPTG when OD6oo
reached 0.7- 0.9 and allowed to grow for 5 hrs. post induction. Optical density of
the samples was determined and growth curve was plotted. The desired C-source
was further used during the growth of cultures in the fermentor.
Fermentor Operation in fed batch mode.
After sterilization of the fermentor, the pH of the fermentor media was set to 6.8
by the addition of 4N NaOH I 4N H3P04 and temperature to 28°C. The fermentor
was started in batch phase with a working volume of 6L. Following inoculation
the DO began to fall. As soon as it touched 20% level, it was controlled not to go
below by increase in agitation automatically. When the C-source in the batch
medium was consumed (indicated by DO rise), the feed was started by the
computer at a flow rate determined by the following equation corresponding to a
set value of specific growth rate 0.16 h-1:
F(t)=[IJ/YXS + m] XO VO [exp{IJ (TFB-TR)}] /SO
where:
F(t) Instantaneous feed flow rate, 1/h
ll Set value of specific growth rate, 1/h
Y xs Growth yield coefficient, g cell/ g C
m maintenance coefficient, g C/ g cell/ h
Xo dry cell mass at the end of batch phase, gil
Vo culture volume at the end of batch phase, I
TFB time elapsed since inoculation, h
T R Batch phase duration, h
t fed batch culture time (=T FB - T B), h
S0 Concentration of the carbon substrate in feed, g/1
The feed medium was 4 litre. Concurrent with the feed addition the DO started to
go down slowly. It was controlled above 20% by automatic increase in agitation
(250 - 550 rpm) and aeration from 0.5 to 1.5 vvm. When air and agitation reached
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upper limits, air was mixed with oxygen (0 - 50%). The pH of the media was
controlled at pH 6.8 by automatic addition of 4N H3P04 in the batch phase.
Thereafter, it was controlled by automatic addition of 10 % malic acid. A second
dose of antibiotics was added to the culture after 24 hrs. At 25 hr., malic acid was
removed and 4N H3P04 was continued for the pH control. The cultures were
induced by 0.1 mM IPTG (5 ml of 1 M stock) at 27 hr. As soon as the feed
finished, the culture was harvested via a heat exchanger to an outlet temperature
of 10- 14oc.
Assay Procedures
(a) Dry Cell Weight
A 10 ml sample was centrifuged (10000 rpm, 10 min). The residue was
transferred to preweighed aluminium cups and dried over night at 80°C in an oven
to a constant weight.
(b) Cell OD
The culture sample was suitably diluted in normal saline in the range
0.1 <OD<0.4. The optical density was promptly read at 600 nm as the cells have a
tendency to settle down.
(c) Protein purification
The pellet from 1 litre of high density culture was resuspended in 200 ml of 50
mM Na-phosphate (pH 7.8) and 300 mM NaCl buffer. Cells were sonicated at
4°C (1-min bursts, 2 min of cooling, 200-300 W) for ten cycles. Apart from
PMSF (1 mM) various other protease inhibitors (Protease inhibitors kit from
Boehringer Mannheim) were added prior to sonication. The lysate was
centrifuged at 10,000 x g for 30 minutes. The supernatant was passed through 20
ml ofNi-NTA slurry. The resin was washed with 50 mM Na-phosphate (pH 6.0)
and 500 mM NaCl buffer. Protein was eluted with a linear gradient of 50 ml each
of 0 and 500 mM imidazole chloride in 50 mM Na-phosphate (pH 7.0), 300 mM
NaCl and 20 % Glycerol. Fractions containing rLF were pooled and dialyzed
against T10E5 (10 mM Tris and 5 mM EDTA [pH 8.0]) overnight. The dialyzed
74
protein was loaded onto a 10 ml Mono-Q (Pharmacia) anion exchange column.
The protein was eluted with a linear gradient of 0 to 500 mM NaCI in T wEs
buffer. The purified LF was dialyzed against 10 mM HEPES pH 7.0 containing
50 mM NaCl and was frozen at -70°C in aliquots.
(d) Quantitation of LF
The fold purification of LF at different column stages was determined by
calculating the amount of protein required to kill 50 % of J774A.1 cells (EC5o)
when incubated with PA (1 J..tg/ml) at 37°C. The protein was measured by the
method of Lowry et al. (1962) as described earlier.
Results
Effect of temperature on recombinant protein production
It is well known that cultivation of recombinant E. coli strains, in fed-batch mode
leading to high cell density in bioreactors, requires very high oxygen transfer rates
to support the growth and to prevent channelling of C-source via anaerobic route
to acetate formation (Korz et al., 1995). Dissolved oxygen (DO) level of 20% or
more has been suggested to prevent acetate formation in addition to control of
specific growth rate.
To maintain the DO at 20%, several strategies are reported in the literature (Korz
et al., 1995), such as
(a) DO is controlled with increasing agitation,
(b) when the agitation reaches upper limit, DO is controlled with
increasing aeration,
(c) when the air flow reaches upper limit, oxygen is mixed with air,
(d) when the oxygen enrichment reaches upper limit, DO is controlled by
increasing fermentor's head space pressure, and
75
(e) when the head space pressure reaches upper limit, DO is controlled
with respect to feed rate which forces the specific growth rate to go
down.
In the present work, the step (e) was checked by lowering the temperature without
affecting the yield of recombinant protein. The advantage is that the same
objective could be met more reliably as DO probe output on several occasion may
show considerable fluctuations causing difficulty in feed addition. The culture
was grown at two temperatures (3 7°C & 28°C) on LB medium in shake flask (220
rpm, 6.8 pH). The results show (Fig. 3.1) that though, the culture at 28°C took
longer to come to final OD, the specific yield of recombinant protein (mg/g-cell)
did not change significantly.
Effect of antibiotic concentration
The antibiotics ampicillin and kanamycin have been used earlier as selection
pressure agents at 100 and 25 mglllevel respectively . The effect of using them at
50% of the usual dose was found desirable as it resulted in about 20% increase in
final OD and proportionally higher recombinant protein yields (Fig. 3.1 ). On the
other hand it is very well known that very high selection pressure, particularly in
bioreactors, lead to severe foaming. A batch profile of one of the fermentor runs
where cultures were grown at 28°C in presence of 50 )lg I ml of ampicillin and
12.5 )lg I ml of kanamycin and induced after 6 hrs. of growth is given in the Fig.
3.2
Effect of C-source on the recombinant protein production
The carbon & nitrogen in complex media comes from yeast extract & tryptone. It
is assumed that 52.5 % of yeast extract is protein and that protein has 16.6% N
and about 50% carbon. In the two medium tested, batch medium had carbon
coming from yeast extract and tryptone while feed medium had an additional C
source namely DL-malic acid. Malic acid was selected due to better results over
glucose, maltose and lactose (Fig. 3.3). It was possible to achieve an OD of 1.8
76
s s:::::
0 0 1.0 ...... ro 0 ·;:;; s::::: Q)
Q
~ (.) ...... ...... 0.. 0
Bacterial growth curve
4
3.5
3
--+- 37°C, F antibiotics,S
2.5 __._ 2SOC, F antibiotics, S
--A- 2ffC, H antibiotics, S
-<>- 28°C, H antibiotics, B
2
1.5
1
0.5
0~~~4=~~~~~~~~~~~~~ 0 2 4 6 8
Time (hrs.)
10 12 14 16
Fig. 3.1 Bacterial growth curve at 37°C or 28oC in presence of antibiotics (ampicillin 100 J.lg and kanamycin 25 J.lg per ml- F, ampicillin 50 J.lg and kanamycin 12.5 J.lg per ml- H) in shake flask (S)or in batch fermentor (B)
' .......... ~ 0 -0 0 -
0
-.......... E c 0 0 (0 -0 0
100
90 \\
80
70
60
50
40 L
20
10
0 0
Profile of the batch run in fermentor
I 400
320
240
160
80
0 2 4 6 8 10 12
TIME (h)
Fig. 3.2 Profile from the computer showing DO (%), Temperature, Agitation
(rpm), pH maii1tained with the addition of 4N NaOH I 4N H3P04, OD (lOx),
Induction \Vith 0.1 mM IPTG after 6 hrs.and Harvest during the growth of
recombinant E. coli in a 14L fermentor.
.......... E a. ~ -z 0 1-<C 1-C!J <C
2
1.8
1.6
1.4
s s::
0 1.2 0 \0
~
.£ r/J s:: <!)
'"0
<;; .g 0.8 0.. 0
0.6
0.4
0.2
0 2 4
Bacterial growth curve
6
Time (hrs.)
8
-+-Maltose
-<>-Glucose
---tr- Malic acid
-D- Lactose
10 12
Fig. 3.3 ·Bacterial growth curve at 28oC in presence of antibiotics (ampicillin 50 ~g and kanamycin 12.5 ~g per ml) in modified LB medium alongwith additional C-source in shake flask.
using malic acid as an alternate C-source. The advantage of selecting malic acid
as an alternate carbon source was that the carbon from malic acid enters the
system via the TCA-cycle and as such will not produce undesirable metabolites.
On the other hand, carbon from glucose, lactose and maltose may be metabolized
by different route which results in the formation of growth inhibitory byproducts.
The results would, therefore, be expected to differ in both the cases. In the
fermentor runs, wherever the malic acid was used, it was added by way of pH
control as 10 % solution in distilled water. Inclusion of the malic acid in feed as
neutralized N a-salt did not give better results. A fermentation profile of one of the
runs where the DL-malic acid was added via the pH control is given in Fig. 3.4.
Using this strategy, it was possible to obtain a final OD600 of23 units.
Effect of specific growth rate on the growth and the recombinant protein
production
By using the experimental growth curve values in a three degree polynomial
equation, the fit growth curve can be plotted from which it is possible to
determine the experimental specific growth rate ()l).
where 'a' is the growth coefficients at different time points.
Specific growth rate ()l) for batch phase can be calculated by the equation
)l = 1/ xV.d (xV)/dt where xis the cell mass and Vis the volume.
Since in batch phase , V is constant
)l = 1/x . dx/dt.
Specific growth rate ()l) for fed batch phase can be calculated by the equation
where 'x' is the cell mass at time 't'.
The specific growth .rate during the fed batch mode was calculated (Fig. 3.5).
During the batch, the growth rate was higher which came down to 0.16 per hour
77
Profile of the fed batch run in fermentor
- 100 600 u C) ~ DO • 90 "0 -11. 80
500 ~ w t- 70 - 400 ..J ~ 60 0 - 50 300 -c 0 40 <(
/ TEif - 30 200 -~ -0 20 c pH MA+ 100 -
::I: 10 Q.
c 0 0 0
0 6 12 18 24 30 36
TIME (h)
Fig. 3.4 Profile of the fed batch run in fermentor. Profi le from the
computer showing DO (%), Temperature, Agitation (rpm), pH maintained
with the addition of Malic Acid (MA+) and removal of Malic acid (MA-),
OD, Feed addition / lOml, Induction with 0.1 mM IPTG and Harvest
during the growth of recombinant E. coli in a 14L fermentor.
:; ~
0 T"" -Cl UJ w u.. -........
E c.. .... -z o·· 1-<( 1-(!) <t
Specific growth rate vs time
0.3
0.25 'i:' ~ ..... - 0.2 ~ ... E • .c --- • • ... 0.15 :r; e Of)
<.I t:: 0.1 ·-<.I ~ Cl.
'JJ 0.05
0 2 4 6 8 10 12 14 16 18 20 22
Time (hrs)
Fig. 3.5 Specific growth rate during the fed batch run in fermentor
during the fed batch phase. Experiments were also performed to see the effect of
specific growth rate in the range of 0.12-0.18 h-1 during the growth phase by
appropriately adjusting the feeding rate profile. In all the experiments, the
specific growth rate during induction phase was brought down to 0.1 h-1. The
results are presented in Table 3.1.
Purification of recombinant Lethal factor
The protein was purified from 1 litre of high cell density culture broth. The cells
were sonicated at 4°C in sonication buffer containing PMSF (1 mM) and other
protease inhibitors. Cytosol was passed through Ni-NTA resin. The pH of the
sonication buffer was kept at 7.8 to allow the maximal binding of the fusion
protein to the Ni-NTA slurry. The resin was washed extensively with wash buffer
having pH 6.0. At pH 6.0 most of the impurities and other contaminating host
proteins that bound non-specifically to the Ni-NTA were washed away without
affecting the binding of 6x His-tagged LF. Recombinant LF (rLF) eluted at a
gradient of 100 mM to 250 mM Imidazole chloride. Affinity purified protein
possessed full length rLF and few other bacterial proteins that bound non
specifically to the Ni-NTA resin. These contaminating proteins were removed by
anion exchange chromatography using Mono-Q column on FPLC (Fig. 3.6). The
protein eluted at a gradient of 300-350 mM NaCI. The purified rLF was dialyzed
against 10 mM HEPES buffer containing 50 mM NaCl and stored frozen at -70°C
in aliquots until further use. One litre of the high cell density culture yielded 12.5
mg ofLF. This rLF was 3127 fold purified compared to the cytosolic preparation
(Table 3.2).
Discussion
The importance of recombinant products for both research and commercial use
has inevitably led to a need to increase the volumetric productivity of fermentation
processes to produce these products. Much efforts have been done at the
molecular level to optimize specific protein expression and product yield.
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Table 3.1
Effect of specific growth rate on recombinant protein production
S.No Specific growth ratel-l (h-1) Yield of recombinant protein Growth phase Induction phase (mg/1)
(1 ). 0.12 0.1 7.0
(2). 0.14 0.1 10.5
(3). 0.16 0.1 12.5
(4). 0.18 0.1 12.0
1-l Specific growth rate was maintained by appropriately adjusting the
feeding rate profile.
A 8 c D E M kDa
. •31
~21
Fig. 3.6 Purification of E.coli expressed LF. The proteins were
analyzed on 12 % SDS-PAGE and stained with Coomassie blue. Lane A,
E.coli SG13009 cells expressing the LF gene; Lane B, cytosolic
preparation of cells expressing LF; Lane C, proteins after Ni-NTA affmity
purification; Lane D, protein after passing through Mono-Q column on
FPLC; Lane E, LF purified from B. anthracis and Lane M, molecular
weight standards.
Table 3.2
Purification of LF from Escherichia coli
Fractions Volume Protein Activity Purification (ml) (mg/ml) (ECso)b. (fold)c
Cytosol a 250 160.71 78.184 1 Affinity Purification 15 1.50 0.038 2057 FPLC 4 3.12 0.025 3127
a Cytosol prepared from 1 litre of high density culture.
b EC50 is defined as the concentration of LF (!lg/ml) along with PA (1
llg/ml) required to kill 50 %of the J774A.1 cells. After 3 hrs. of incubation,
viability determined by MTT dye. The results represent the mean of three
experiments.
c Purification fold was determined by dividing EC50 for cytosol with EC50
for fractions obtained from different columns.
Escherichia coli has been widely used as the favourable host for many
recombinant DNA products as the recombinant methodologies of E. coli are very
well developed. Protein expression can be manipulated using different expression
vectors. Transcription of foreign genes in these vectors can be regulated through
the use of appropriate promoter. One can choose, chemically inducible promoters
such as lac, tac or trp promoters to differentiate growth and production phases.
High cell density cultivation of these cultures have been one of the most effective
ways to increase cell density as well as product yields. In fermentor cultivation
the attention is focussed on increasing the volumetric productivity through an
increase in host cell mass. However with high cell density growth there is
formation of unwanted byproducts. These byproducts are partially oxidized
glucose metabolites such as acetic acid, ethanol and lactic acid etc. which have
inhibitory effect on cell growth and productivity (Gleiser et al., 1981). Thus
strategies to attain high cell densities of E. coli have focussed primarily on
minimizing organic acid production.
Acetate and other byproducts are produced under anaerobic conditions in the
fermentor or when there are excess of nutrients as a result of which the specific
growth rate exceeds the growth rate at which acetate is formed (Zabriskie et al.,
1986). To address these problems, fed batch fermentation have been employed to
obtain high cell densities while minimizing acetate formation. In the conventional
batch process, the production phase is short, due to the depletion of the carbon
energy source; the subsequent cell autolysis is rapid and severe. Therefore, after
transition from growth to synthesizing phase, it is important to maintain a
concentration of the carbon energy source where the microorganisms are semi
starved but where enzyme activity for synthesis is the highest. The carbon source
feeding is controlled to minimize or delay acetate formation by limiting its
concentration and subsequently the specific growth rate. Furthermore, the feeding
is controlled so that the dissolved oxygen concentration does not become limiting
and the aerobic cultivations operate within the limits of the system (Zabriskie et
al., 1986; 1987). Exponential feeding which results in a constant specific growth
rate below the critical growth rate at which acetate is formed was used in the
present work. In the earlier chapter, we described the purification of recombinant
79
lethal factor by growth of E. coli cultures in shake flask. In the present studies,
the strain producing recombinant protein was bioprocess optimized to enhance the
yields of the recombinant protein. Cultures were grown in the presence of half
antibiotic concentrations and at 28°C. There was about 20 % increase in the
optical density of 14 hrs. grown culture. Various C- sources such as glucose,
lactose, maltose and DL malic acid were tried. Glucose leads to the formation of
ethanol and other byproducts even in the presence of sufficient dissolved oxygen
(DO) if an excess of sugar is present in the culture medium. These byproducts are
the main cause of low cell and product yields (Han et al., 1993). The carbon from
glucose, maltose and lactose enters the metabolic pathway via glycolysis which
results in the formation of pyruvate which can further form ethanol, while the
carbon from the malic acid enters the system via the TCA cycle and thus avoiding
the production of undesirable metabolites. Malic acid (1 0 % ) was added in the
feed medium by way of pH control. Malic acid was also incorporated in the feed
medium (30 g /1) and was added to the culture along with the feed. However, the
final OD achieved was 12.6 which was much less than that achieved when added
by way of pH control.
In the early log phase, cells grew at the specific growth rate of 0.16 h-1 compared
to 0.1 h-1 for the late log phase induced cultures. This pause in growth may be an
indication of the increased metabolic burden placed on the cell due to recombinant
protein synthesis.
We could successfully cultivate recombinant E. coli to an optical density of 23
units with 35 grams of dry cell weight per litre of the culture. The protein was
purified using the Ni-NTA chromatography and anion exchange chromatography
using a Mono-Q column on FPLC. In the earlier purification procedure as
described in chapter 2, size exclusion chromatography was used to remove the
degraded protein products. However, in the present studies a set of protease
inhibitors was used to prevent the proteolytic degradation by different proteases.
This enabled to purify the recombinant lethal factor to homogeneity in two steps
with a purification fold of 3127 as compared to the cytosolic protein. It was
possible to purify 12.5 mg 11 of the fed batch culture as compared to 1.5 mg 11 of
the culture in the shake flask.
To conclude it is demonstrated that high cell densities are obtainable for this
80
expression system with concomitant recombinant protein expression. Use of DL
malic acid as an alternate C- source is advantageous. Lowering of temperature
and maintaining the specific growth rate at two different levels improved overall
product yield. Present work is an effort to harness the capabilities of the miqro
organisms to produce the recombinant proteins for further use in industry,
medicine, agriculure and research etc.
81