Biomedical Engineering Reference
In-Depth Information
of cultivation and the composition of the nutrient medium. All further stages of isolation and
purification of recombinant TBI protein accumulated in inclusion bodies depend on the yield
of cells. Thus, when developing cultivation conditions, it is necessary to find an optimal
combination of parameters that provide a stable and high yield of the target protein.
The goal of the study is to optimize the cultivation process that ensures the yield of
biomass with a high content of TBI protein.
M
ATERIALS AND
M
ETHODS
Recombinant
E.coli
JM 103/pTBI producing strain was used for optimization of the
cultivation process.
Experimental multicomponent TB and TB plus media were used as nutrient media. YT
medium served as a control [3, 4, 9]. TB medium consisted of tryptone, 12.0 g/l; yeast extract,
12.0 g/l; glycerol, 4.0 g/l; KH
2
PO
4
, 2.31 g/l and K
2
HPO
4
, 12.5 g/l, ampicillin, 200 µg/ml. TB
plus medium was supplemented with a trace element solution.
Cultivation of
E.coli
JM 103/pTBI recombinant strain was carried out in 750 ml
Erlenmeyer flasks containing 100 ml of the nutrient medium. The content of the flasks was
shaken at 180 rpm in a New Brunswick E-25 incubator.
The dose of inoculate was 1 % of the nutrient medium volume. In the process of
cultivation, nalidixic acid was added as a protein biosynthesis inducer. Upon completion of
the fermentation process, biomass was separated by centrifugation (8000 rpm/min, 15 min).
Optical density was measured by the spectrophotometer at 550 nm. The content of
recombinant TBI protein in biomass was analyzed by electrophoresis in 12 % PAAG
followed by densitometric scanning [3, 5].
Fermentation was conducted in a laboratory fermenter with a working volume of 10 liters
(“Ultraferm”, LKB, Sweden) equipped with the systems of measurement of temperature,
stirrer speed and aeration rate. The dose of the inoculate was 5 % of the nutrient medium
volume and fermentation was carried out for 6-7 h.
Fermentation conditions: 37 °C, 200 rpm/min. Aeration conditions: air was passed at a
rate of 1 vvm (volume of air per unit volume of medium per minute) from the beginning of
fermentation to the addition of the inducer. Then the air flow rate was increased up to 1.7
vvm.
Isolation and purification of recombinant TBI protein was carried out as follows: the cells
were resuspended with buffer (10 mM Tris/HCl, pH 8.0, 1 : 4 v/v) and degraded by
ultrasound.
ДЕАЕ-cellulose ДЕ-52 (Whatman, England) was used for chromatographic purification
of TBI protein.
SDS/PAGE electrophoresis was performed with 12 % (w/v) gel by the Laemmli method
[6].
Quantitative analyses were carried out using a computer program «Gel-pro analyzer, Ver.
3.1ﾻ (Media Cybernetics Inc. США). The yield of TBI protein was calculated in percents
from the initial content of the total protein.
The content of bacterial endotoxin in the purified protein was evaluated by the LAL test.
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