Hi all.
I want to high the amount of the Heat shock chaperones concentration. I read one option is to do a Heat shock step before the protein induction. Anyone know How could I do?
Thank you.
Pilot screening of protein expression in presence of different concentration of ethanol
1.
Pick a single colony from the transformed LB agar plate or take 20 μL of glycerol stock and inoculate into 5 mL of LB broth containing appropriate antibiotics according to the vector construct and host cells.
2.
Incubate the culture at 37 °C for overnight (14–16 h) at 180 rpm with continuous shaking as starter cultures.
3.
Next day take five culture vials and name them as:
•
Control (un-induced).
•
Induced in the absence of ethanol.
•
Induced in the presence of 1% ethanol.
•
Induced in the presence of 2% ethanol.
•
Induced in the presence of 3% ethanol.
4.
Inoculate five vials (each containing 5 mL LB medium with appropriate antibiotics) with 20 μL of the overnight grown starter cultures.
5.
At the time of inoculation add 1%, 2% and 3% ethanol to the LB medium and grow at 37 °C for a few hours (approx. 3–4 h.) with vigorous shaking, until the OD600 reaches 0.5–0.6.
Note: Ethanol should be used in v/v ratio. We have optimized the optimum ethanol concentration and it was observed that 3% ethanol (v/v) gives the maximum enhancement in protein expression. In the presence of more than >5% ethanol cell growth was found to be inhibited.
6.
Induce the protein expression by adding IPTG to a final concentration of 1.0 mM.
Note:
•
Do not add IPTG to the culture which will serve as a non-induced control.
•
Minimal IPTG concentration should be optimized in small scale of culture before proceeding to the mass culture.
•
The optimal growth time for TB (Terrific broth) is different from LB (Luria broth) In case of TB OD600 should be more than 1.0–1.5 before IPTG induction.
•
In case of auto-induction media the control should be normal LB not the auto-induction media. There is no need to observe the OD, because it does not need the IPTG induction.
7.
Grow the cultures for an additional 4–5 h at 37 °C with continuous shaking.
8.
Harvest 1 mL of cells from each culture vial by centrifugation for 1 min at 12,000 rpm. Discard the supernatants (remaining media).
9.
Re-suspend the cells in 60 μL of buffer (Tris or phosphate or PBS, pH-8.0), 20 μL of 10% SDS & 20 μL of 5X SDS loading dye and lyse by mixing or pipetting.
10.
Boil the samples in 100 °C for 5–10 min (Vortex the samples in between).
11.
Load the sample in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and analyze. Screening through pilot experiment is important, which will give the clear picture about the level of over-expression of the protein in the presence of 1%, 2% and 3% ethanol. This will also provide the opportunity to compare the over expression of recombinant protein in the presence and absence of ethanol. SDS-PAGE analysis will support the increase fold of expression with increased percentage of ethanol.
Note: Pilot screening can be done at low temperature ranging from 16 °C to 23 °C for 20–22 h after induction with IPTG, depending upon the level of expression and solubility screening of the recombinant proteins. In case of auto-induction media incubate the culture until the OD600 reach 0.4–0.6. It is observed that some proteins give higher fold of expression in low temperature. This screening can also provide a clear idea about the level of expression of the protein, which can be helpful for further mass culture. For mass culture setup the experiment according to the same ratio of pilot experiment.
Dear
I add you also others contributing sources.
http://home.sandiego.edu/~josephprovost/E.%20Coli%20protein%20expression%20protocol.pdf
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3063
http://www.ncbi.nlm.nih.gov/pubmed/18428660
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771296/
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