In this case, prokaryotic expression system was used. The
recombinant plasmid was transformed to E.coli and the protein expression was
analyzed by SDS-PAGE. The Ni-column affinity purification method was used to
get target protein.
Any organization or individual may not reprint or
misappropriate without the permission of Elabscience. The offender will be
investigated for legal responsibility.
Order analysis
Analyze the hydrophilic, surface antigenicity, etc
according to protein sequence customer provided, choose the region to be
expressed. Optimize the codon of chosen region and develop a proposal with
highest expression rate.
Experiment process
1. Plasmid construction
Synthesize the optimized gene. Digest the pET28a vector by Ncol
enzyme and Xhol enzyme and link the gene to it, then select positive clones to
sequence.
2. Transform pET28a-XXX to E.coli
a. Add 1 μL of plasmid to 100 μL of competent cell suspension,
gently shake and mix fully, keep on ice bath for 30 min.
b. Heat shock on 42℃ water bath for 90 s,
keep on ice for 3 min, then add 800 μL of LB and keep at 37℃ for 45 min.
c. Centrifuge at 12,000 g for 1 min, pipette 800 μL of supernatant, mix the remaining supernatant and bacteria again.
d. Spread the remaining bacterium solution to
selective solid medium containing kanamycin. Put the medium upside down the in
37℃ constant temperature incubator for 16 h
after the bacterium solution was fully absorbed.
3. Expression of the recombinant protein
a. Select individual bacterial colonies containing
recombinant plasmid to inoculate in 5 mL of LB(contains kanamycin) medium,
culture at 37℃ overnight.
b. Take 1 sterile preservative tube containing
50% of glycerinum, add 700 μL of bacteria solution and reverse it to mix up.
Store the tube at -80℃.
c. Pipette 500 μL of bacteria solution to inoculate in
50 mL of LB medium, culture at 37℃ till OD600=0.6-0.8.
d. Pipette 1 mL of uninduced bacteria solution to sterile
2 mL centrifuge tube as control. Add IPTG to the remaining bacteria solution to
reach a final concentration 0.5 mM. Culture the bacteria solution at 30℃ for 5 h.
e. Pipette 1 mL of induced bacteria solution and
uninduced bacteria solution respectively to centrifuge, re-dissolve with 500 μL of binding buffer. Analyze the protein by SDS-PAGE.
Protein expression identification
4. Purify the
recombinant protein
a. Collect 300 mL
of bacteria culture, centrifuge at 5000 rpm for 5 min. Re-dissolve with half
binding buffer.
b. Broken the
bacteria by ultrasonication at 450 W for 30 min.
c. Centrifuge at 12,000×g
for 10 min.
d. Re-dissolve the precipitation with one-tenth
wash solution.
e. Centrifuge at 12,000 ×g for 10 min, abandon the supernatant.
f. Wash repetitively
for 3 times.
g. The purity of inclusion bodies was over 80% by SDS-PAGE
determination.
5. Renature inclusion bodies
a. Dissolve the inclusion bodies in solution
buffer.
b. Centrifuge at 12,000 × g for 10 min, collect the supernatant.
c. Add the supernatant to dialysis bag with appropriate pore
diameter, put it to refolding buffer with 20 times volume, keep at 4℃ for 16 h.
d. Centrifuge at 12,000 × g for 10 min, collect the supernatant.
Determine protein concentration by BCA method and protein purity by 15%
SDS-PAGE.
e. Freeze-dry protein: add protective agent to
sample and freeze-try.
Renatured protein identification
In this case, prokaryotic expression system was used. The
recombinant plasmid was transformed to E.coli and the protein expression was
analyzed by SDS-PAGE. The Ni-column affinity purification method was used to
get target protein.
Any organization or individual may not reprint or
misappropriate without the permission of Elabscience. The offender will be
investigated for legal responsibility.
Order analysis
Analyze the hydrophilic, surface antigenicity, etc
according to protein sequence customer provided, choose the region to be
expressed. Optimize the codon of chosen region and develop a proposal with
highest expression rate.
Experiment process
1. Plasmid construction
Synthesize the optimized gene. Digest the pET28a vector by Ncol
enzyme and Xhol enzyme and link the gene to it, then select positive clones to
sequence.
2. Transform pET28a-XXX to E.coli
a. Add 1 μL of plasmid to 100 μL of competent cell
suspension, gently shake and mix fully, keep on ice bath for 30 min.
b. Heat shock on 42℃ water bath for 90 s,
keep on ice for 3 min, then add 800 μL of LB and keep at 37℃ for 45 min.
c. Centrifuge at 12,000 g for 1 min, pipette 800 μL of supernatant, mix the remaining supernatant and bacteria again.
d. Spread the remaining bacterium solution to
selective solid medium containing kanamycin. Put the medium upside down the in
37℃ constant temperature incubator for 16 h
after the bacterium solution was fully absorbed.
3. Expression of the recombinant protein
a. Select individual bacterial colonies containing
recombinant plasmid to inoculate in 5 mL of LB (contains kanamycin) medium,
culture at 37℃ overnight.
b. Take 1 sterile preservative tube containing
50% of glycerinum, add 700uL of bacteria solution and reverse it to mix up.
Store the tube at -80℃.
c. Pipette 500 μL of bacteria solution to inoculate in
50 mL of LB medium, culture at 37℃ till OD600=0.6-0.8.
d. Pipette 1 mL of uninduced bacteria solution to sterile
2 mL centrifuge tube as control. Add IPTG to the remaining bacteria solution to
reach a final concentration 0.5 mM. Culture the bacteria solution at 30℃ for 5 h.
e. Pipette 1 mL of induced bacteria solution and
uninduced bacteria solution respectively to centrifuge, re-dissolve with 500 μL of binding buffer. Analyze the protein by SDS-PAGE.
Protein expression identification
4. Purify the recombinant protein
a. Collect 300 mL of bacteria culture, centrifuge at
5000 rpm for 5 min. Re-dissolve with half binding buffer.
b. Broken the bacteria by ultrasonication at 450 W for 30 min.
c. Centrifuge at 12,000 ×g for 10 min.
d. Re-dissolve the precipitation with one-tenth
wash solution.
e. Centrifuge at 12,000 ×g for 10 min,
abandon the supernatant.
f. Wash repetitively for 3 times.
g. The purity of inclusion bodies was over 80% by SDS-PAGE
determination.
5. Renature inclusion bodies
a. Dissolve the inclusion bodies in solution
buffer.
b. Centrifuge at 12,000 ×g for 10 min,
collect the supernatant.
c. Add the supernatant to dialysis bag with appropriate pore
diameter, put it to refolding buffer with 20 times volume, keep at 4℃ for 16 h.
d. Centrifuge at 12,000 ×g for 10 min,
collect the supernatant. Determine protein concentration by BCA method and
protein purity by 15% SDS-PAGE.
e. Freeze-dry protein: add protective agent to
sample and freeze-try.
Renatured protein identification
In this case, expression vector was provided by
customer. The protein expression was analyzed by SDS-PAGE and the Ni-column
affinity purification method was used to get target protein.
Any organization or individual may not reprint or
misappropriate without the permission of Elabscience. The offender will be
investigated for legal responsibility.
Order analysis
As the target protein was fused with GST tag, we purified
the protein by Glutathione Sepharose and removed the tag by enzyme digestion,
then purified again to get target protein.
Experiment process
1. Expression of the recombinant protein
a. Pipette 10 μL of bacteria solution, spread on 5 mL
of LB medium. Culture at 37℃ overnight.
b. Take 1 sterile preservative tube containing
400 μL of 50% glycerinum, add 700 μL of bacteria solution and reverse it to mix
up. Store the tube at -80℃.
c. Inoculate the remaining bacteria solution in
300 mL of LB medium, culture at 37 ℃ till OD600=0.6-0.8.
d. Add IPTG to a final concentration of 0.5 mM, Culture
the induced and uninduced bacteria solution at 30℃ for 5h.
e. Collect uninduced and induced bacteria solution
to analyze with 12% SDS-PAGE.
Protein expression identification
2. Purify the recombinant protein
a. Gently turn the Glutathione Sepharose
upside down.
b. Add 3 mL of resin to 10 mL of purification
column. Keep the resin precipitating by gravity for 5~10 min.
c. Wash the
column with sterile distilled water(the volume is 3~5 times of column).
d. Wash the
column with binding buffer containing 5 mM of DTT (the volume is 3~5 times of
column).
e. Load
sample (target protein less than 20 mg in the sample). Store
the effluent at 4 ℃, analyze by
SDS-PAGE.
f. Wash the
column with wash buffer (the volume is 3~5 times of column) till the effluent
was identified no protein in by G250. Store the effluent at 4℃, analyze by
SDS-PAGE.
g. Elute target
protein with elution buffer.
h. Collect 1.5 mL
of eluent in each tube till no protein was eluted off.
i. Add the protein
solution to dialysis bag, dialyze with PBS (pH=7.4) overnight.
3. Enzyme digest
a. Add thrombin to protein sample. Enzyme digest at
37℃ for 20 min.
b. Load the enzyme-digested product as
outlined above. Collect the effluent.
c. Analyze by 12% SDS-PAGE.
Enzyme digested product identification
In this case, prokaryotic expression system was used. The
recombinant plasmid was transformed to E.coli and the protein expression was
analyzed by SDS-PAGE. The Ni-column affinity purification method was used to
get target protein.
Any organization or individual may not reprint or
misappropriate without the permission of Elabscience. The offender will be
investigated for legal responsibility.
Order analysis
Analyze the hydrophilic, surface antigenicity, etc
according to protein sequence customer provided, choose the region to be
expressed. Optimize the codon of chosen region and develop a proposal with
highest expression rate.
Experiment process
1. Plasmid construction
Synthesize the optimized gene. Digest the pET28a vector by Ncol
enzyme and Xhol enzyme and link the gene to it, then select positive clones to
sequence.
Plasmid profile
Part of
sequencing result
2. Transform pET28a-XXX to E.coli
a. Add 1 μL of plasmid to 100 μL of competent cell
suspension, gently shake and mix fully, keep on ice bath for 30 min.
b. Heat shock on 42℃ water bath for 90 s,
keep on ice for 3 min, then add 800 μL of LB and keep at 37℃ for 45 min.
c. Centrifuge at 12,000 g for 1 min, pipette 800 μL of supernatant, mix the remaining supernatant and bacteria again.
d. Spread the remaining bacterium solution to
selective solid medium containing kanamycin. Put the medium upside down the in
37℃ constant temperature incubator for 16 h
after the bacterium solution was fully absorbed.
3. Expression of the recombinant protein
a. Select individual bacterial colonies containing
recombinant plasmid to inoculate in 5 mL of LB (contains kanamycin) medium,
culture at 37℃ overnight.
b. Take 1 sterile preservative tube containing
50% of glycerinum, add 700 μL of bacteria solution and reverse it to mix up.
Store the tube at -80℃.
c. Pipette 500 μL of bacteria solution to inoculate in
50mL of LB medium, culture at 37℃till OD600=0.6-0.8.
d. Pipette 1 mL of uninduced bacteria solution to sterile
2 mL centrifuge tube as control. Add IPTG to the remaining bacteria solution to
reach a final concentration 0.5 mM. Culture the bacteria solution at 30℃ for 5 h.
e. Pipette 1 mL of induced
bacteria solution and uninduced bacteria solution respectively to centrifuge, re-dissolve
with 500 μL of binding buffer. Analyze the protein by SDS-PAGE.
Protein expression identification
f. SDS-PAGE with uninduced and induced bacteria
solution, transfer for 1.5 h to membrane, block with milk for 1 h, add anti-his-tag
antibody and incubate at room temperature for 1 h. Wash with TBST 3 times, add
secondary antibody and incubate for 1 h. Wash with TBST 3 times, then develop.
His-tag WB identification
4. Purify recombinant protein
a. Collect 300 mL of bacterial solution, centrifuge
at 5000 rpm for 5 min. Re-dissolve it with one tenth volume of binding buffer.
b. Broken the bacteria by ultrasonication at 450 W for 30 min.
c. Centrifuge at 12,000 ×g for 10 min, transfer the supernatant to
another container. Repeat this step.
d. Take 40 μL of lysis and 40 μL of supernatant
respectively to analyze by SDS-PAGE.
e. Add 3mL of resin to 10 mL of purification column.
Keep the resin precipitating by gravity for 5~10 min.
f. Wash the column with sterile distilled water (the
volume is 3~5 times of column).
g. Wash the column with binding buffer (the volume
is 3~5 times of column).
h. Load sample (target protein less than 20 mg in the
sample).Store the effluent
at 4℃, analyze by SDS-PAGE.
i. Wash the column with wash buffer (the volume is
3~5 times of column). Store the effluent at 4℃, take sample to
analyze by SDS-PAGE.
j. Elute target protein with elution buffer.
k. Collect 0.5 mL of eluent in each tube till no
protein was eluted off.
l. Add the protein solution to dialysis bag, dialyze
with PBS(pH=7.4) overnight.
Purified protein identification
