Hi there,
I´ve tried to purify a GST-tagged protein using IPTG and GST-Sepharose beads. Although I have used several bacterial volumes (50.100.200 ml), differents IPTG concentrations (0.5 and 0.2 mM) and different buffers I haven´t obtained satisfactory results. On the other hand, my control (GST empty vector) is properly expressed and purification is successful. I have also controlled the D.O using 0.6 and 0.7 values. Does anyone recommend me to increase the IPTG concentration? (I have heard that someone used 200 mM of IPTG).
I would be extremely grateful to anyone who provides me with a piece of advice.
Thank you a lot in advance
Check your construct. I don't think that less IPTG will end up not having protein, GST is quite robust. Are you sure that your beads are clean ... Usually, high pH and low pH buffer wash will clean them.
Hi Alejandro,
Did you check over-expression of your protein before proceeding to purification step? Also did you do plasmid sequencing of your construct to check if the gene of interest is in-frame or not, if there is any stop codon or something?
If you have the sequence of your construct and primers, you may try Serial Cloner software which virtually translate your protein from the sequence, get no. of amino acids, mol wt., pI etc details. If plasmid sequence goes well, then you may do an extra step of checking the over-expression in small scale such as 5ml. of LB culture with a gradient of IPTG concentration. Comparing set of samples with and without IPTG addition is expected to show presence of over-expressed protein. If this step also goes well, then you may check with your beads. Did you run your beads previously just in case the protein got precipitated in the beads!!
Less is sometimes more with IPTG, many years ago I only saw soluble IL7 expression with 100uM! Also cold temps sometimes decreases the target proteins toxicity, 30C still gives OK growth but was essential to get a glucosidase to be soluble more recently. If its really a problem protein you could look into a Studier autoinduction protocol; the same glucosidase expressed at >1g/L (soluble!) in shake flasks left to grow for 4 days at room temp in the Studier media, Good luck!
Seema Nath Thanks a lot for your reply !. Plasmid sequencing proved that the protein is in frame and beads were also runned. However, (contrary to the GST empty vector control) I haven´t seen any overexpression of this protein after IPTG induction. I will carry out the IPTG gradient and check weather this protein is expressed or not
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