UNIVERSITETET I LINKÖPING
Proteinkemi
Kemiavdelningen
2012-01-17
Expression, production and purification of Fatty
acid binding protein (FABP) from desert ant
Cataglyphis fortis
Production of FABP in E. coli
1. Introduction
In this laboratory experiment, a protein, fatty acid binding protein, FABP, from the desert ant
Cataglyphis fortis, is produced by E. coli bacteria. These bacteria contain a plasmid, in which
the gene for FABP is inserted. This plasmid is also an expression vector, which means that
you can turn on transcription of the FABP gene with an inducer. A repressor molecule is
initially bound to the operator region of the lac promoter. As an Inducer (IPTG, isopropyl-D-tiogalaktopyranosid) is added it binds to the repressor molecule, thus loosening the operator
gene, leading to RNA polymerase can initiate transcription of T7 RNA polymerase. T7 RNA
polymerase binds tightly to the T7 promoter, resulting in efficient transcription of the FABP.
Plasmid also contains a gene, which codes for antibiotic resistance. This means that one can
distinguish between bacteria that have the plasmid from those that lack this, by adding
antibiotics to the culture medium. In this way, only the bacteria containing the plasmid are
able to grow.
The produced FABP will be located in E. coli cytoplasm. This means that you have to
destroy bacterial cell walls, to access the FABP. This is done by means of sonication, ie break
the cell membrane by ultrasound.
When the cell wall is separated from the cytoplasm by centrifugation, to obtain a solution
containing only cytoplasm. The cytoplasm is now containing FABP with approximately 2000
other proteins. By using a His-Tag-specific purification method one can separate the FABP
from all the other proteins in a single purification step (His-Tag chromatography).
Expression of the cloned and mutated protein can be monitored by SDS-PAGE. Therefore,
withdraw samples at different stages of production and purification process for analysis for
SDS-PAGE-lab. Save 50μl solution in eppendorf tubes in a freezer for analysis. You will
choose how many tests and after the operation as a solution to be saved.
Discuss with laborationshandledaren how many samples and at what stages of the production
samples should be withdrawn tests.
Sample preparation
Make following solutions:
LB-Medium
20g/l Trypticase (Peptone)
20 g/l NaCl
10 g/l Yeast Extract
Kanamycinstock (30 mg/ml) 10 ml
Take 300 mg Kanamycin and dissolve in 10 ml dist-H2O. Sterile filtering into autoclaved
Eppendorf tubes ~1.5 ml in each tube. Store the stock solution in freezer and thaw it in room
temperature before use..
IPTG stock (0.5 M ) 10 ml
Take 1.19 g IPTG and dissolve in 10 ml Dist H2O. Sterilfiltrera lösningen genom att trycka
lösningen genom ett sterilfilter ned i sterila Eppendorfrör ~1.5 ml i varje rör. Stammen
förvaras i frys och tinas inför användning i rumstemperatur.
Autoclaving
Autoclave the following materials:
1.5 l LB in 5 l Erlenmeyer flask
2 x 250 ml bottle
O / N culture flasks with 15 ml
LB-medium should be autoclaved and autoclaving time is calculated by using the table in the
Autoclaving room.
Inoculation of bacteria to a small culture:
After the autoclave when no solution has cooled, add 30 30 g/ml kanamycin (1000 times
dilution) for overnight culture flask (200ml flask) containing 15 ml LB medium.
Thaw bacteria strains on the ice. Add the appropriate amount of bacteria, approximately 50200 l. Grow at 37 C overnight.
Inoculation of bacteria to a large culture:
Add 30 g/ml Kanamycin (1000 times dilution) to large flasks (5 l ) containing 1,5 l LBmedium.
Transfer all solution from O/N culture (15 ml) to the large 1,5-liter LB-solution
Grow at 37 C with shaking.
Common continuation:
Then follow the growth by measuring absorbance at 660 nm, which reflects cell density. This
is done by absorbing LB medium with a sterile pipette (~3-5 ml). Measured in plastic
cuvettew, using distilled-H2O as a reference.
When absorbance is approximately 0.8 added 1.5 ml of the Inducer, IPTG.
to induce the production of Fatty acid binding protein (FABP) in E. coli bacteria. After this
addition the growth continue at room temperature overnight.
The growth is halted after 12-15 hrs. Bacterial solution is centrifuged at 3200 rpm for 20 min
(4 C) in large centrifuge bottles. In this way one can separate the bacterial cells from the
culture medium. The supernatant is poured gently back into the flask and then autoclaved
before being discarded. The pellets consisting of bacteria suspended in a small volume (max.
30 ml) resuspension buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mm Imidazole, pH 8) to NiNTA chromatography (His-Tag purification) for breaking capture of bacterial cells with by
sonication.
Sonication (ultrasound treatment):
Sonication is often used to break the cell membranes, with ultrasound (typically 20-50 kHz) is
generated electronically and the mechanical energy is transferred to the sample via a metal
probe that oscillates at high frequency, resulting in limited areas with high pressure. This
leads to cavitation and compression in the solution, which ultimately makes the cells are
broken up and the protein content comes out of solution. Sonication is 6x10 sec with 10 sec
intervals on a sample of up to 30 ml. The samples are kept on ice during the process until the
chromatography is done.
Qiagen Ni-NTA chromatography
Solutions:
Resuspension buffer (20 mM Tris-HCl, 150 mM NaCl, 5mM Imidazole, pH 8)
Washing buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM Imidazole, pH 8)
Elution bufferA (20 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, pH 8)
Elution buffer B (20 mM Tris-HCl, 150 mM NaCl, 500 mM Imidazole, pH 8)
Equilibrate the column with 10 ml resuspension buffer
Load the sample (1-35 ml). The flow should be approximately 1 ml/min. Collect the runthrough in a flask or a beaker.
Wash with 4 ml wash buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM Imidazole, pH 8).
Collect the run-through in a flask or a beaker.
Elute with 3 ml elution buffer A (20 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, pH 8)
Load 3 ml elution buffer B (20 mM Tris-HCl, 150 mM NaCl, 500 mM Imidazole, pH 8).
Collect fractions in Eppendorf tubes, 1 ml in each fraction)
Pool the fraktions containing FABP and dialyze the sample
After use: Wash the gel with 30 ml 0.5 M NaOH. store in 30 % etanol.
SDS-Polyacrylamidegelelectrophoresis (SDS-PAGE)
1. Introduction
After FABP purified by affinity chromatography, the purity is studied using SDS-PAGE. In
addition, the purity and expression of FABP studied on samples withdrawn at different
moments during the production process. It is the most common method to monitor a
purification of a protein and determine the purity of the protein is. This method separates
proteins according to their size. By SDS (sodium dodecylsulfate), which is negatively
charged, added to the sample, the protein carries a negative charge, which is directly
proportional to protein size. This is done by SDS binds to the protein. Polyacrylamide gel
consisting of a network that proteins have to walk through. Size of the network depends on
the ratio of acrylamide and bis-acrylamide. These proportions are chosen so that the proteins
they want to study separate from each other on the gel. It also includes a number of reference
proteins of known molecular weight in its analysis. These run separately on the same gel, so
that from these may appreciate their own sample's molecular weight. By allocating the
logarithm of molecular weights of reference proteins against migrating distance a straight line
is obtained. It is then easy to estimate the molecular weight of the sample using the reference
graph.
Note! To this lab pre-made polymerized gel plates are used
Sample Preparation:
Sample: Appropriate sample quantity to be applied is approximately 10 g of protein, the
maximum volume of a sample well is approximately 35 l. First calculate the volume of
sample you will receive. The sample is mixed then with 10 l sample cocktail, and 1 l mercaptoethanol. Add 5 l of bromphenol blue (BFB). The mixture is heated in boiling water
for 2-3 min. Then applied to the sample in the test wells.
Reference: 5 l premixed reference solution, 10 l sample cocktail, 5 l BFB and 1 l mercaptoethanol mixed. This is then treated as above.
electrophoresis
Mount the gel cassette . Dilute electrode buffer 5 times with distilled-H2O to a final volume
of 850 ml and pour it into electrophoresis chamber (in the "inner" and "outer" space).
Run at 50 V through concentration gel and then increase to 200 V. Stop the run when the
blue band has migrated to the end of the gel.
Fixation, staining and destaining:
1. Remove gel from glass plates
2. Place the gel in water so that it is well covered
3. Heat in the microwave so that it almost boils. It must not boil, which threaten to destroy
the gel. Take about so much that it takes 40 s to heat the water.
4. Pour off the water without pouring the gel.
5. Repeat steps 2-4 three more times
6. Pour Safestain over the gel so that it covered. Heat in microwave until it almost boils.
7. Stain in 30 min
8. Bleach in water
Photography of the gel:
The gel is washed in distilled water, H2O, and then stored in 20% (v / v) ethanol before the
shooting.