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

68

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

69

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:

70

. ---------------------------------------------------------------------------------------------------

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)

71

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

72

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

73

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.

78

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