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Protein that used to express really well stopped expressing.

Protein expression GST tagged proteins

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2 replies to this topic

#1 bpuq



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Posted 14 February 2016 - 04:38 PM



I have expressing and purifying my GST tagged protein successfully until a few weeks ago. I used to use my glycerol stock for the expression instead of freshly transforming everytime, and it worked really well. Now it stopped expressing and i tried transforming into DH5alpha and then into Rosetta (DE3). But the expression is still not there. I tried the same expression conditions that used to work for me before, and also tried decreasing the IPTG concentration to 0.2 mM and tried decreasing the temperature too. Still not helping. Any suggestions?


Thank you in advance.



#2 phage434



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Posted 15 February 2016 - 05:16 AM

Perhaps the cells you are transforming into have changed. Are you sure you are transforming into the same strain? I would also immediately sequence the plasmid to make sure I was transforming the construct I expected. Where did the plasmid come from? Is it the original plasmid, or was it prepared from the expression strain glycerol?

#3 labtastic



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Posted 15 February 2016 - 06:54 AM

As phage suggested, always do your quality control checks to make sure your are using the strains you think you are using with the plasmid you think you are using.


Assuming these check out, my two most important rules for getting consistent expression every time:


1. Always always always do a fresh transformation into BL21 cells. Never use frozen BL21 glycerol stocks as they can stop expressing even though they remain resistant to the antibiotic. I have seen this many many times. Nobody can explain why this happens but it does. unsure.png


2. Do not express from single BL21 colonies...the myth of needing to select a single BL21 colony for expression is one of the most detrimental dogmas in protein expression protocols. Instead, scrape hundreds of colonies from a freshly grown transformation plate and inoculate starter culture with them. By scrapping hundreds of colonies you guarantee consistent expression every time. Cultures grown from single BL21 colonies can and often do have different levels of expression (I've tested this many times). Sometimes the difference is small, sometimes it is a lot. Do you want to run the risk of expressing from one of these bad colonies? Make it a non-issue by scraping dozens of colonies and getting the population average. Let this starter culture go for 4-5 hours (not overnight), then inoculate into bigger flask(s). Let grow until proper OD, add inducing agent (e.g. IPTG) and express. Harvest when needed (3-4 hours at 37C, overnight at 18C, etc).


Basically, the key steps are (i) fresh transformation, (ii) scraping multiple colonies into your expression culture, and (iii) never letting your bacteria reach stationary phase (this includes starter cultures).


Sure, there are systems and plasmids where you can get away with not doing these things, but by adhering to these you will be guaranteed consistent levels of expression each and every time year after year after year.

Edited by labtastic, 15 February 2016 - 11:18 AM.

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