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Periplasmic Expression - Your Experience...

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#1 DaRQsiDe



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Posted 19 August 2011 - 05:37 PM

Hi all,

I wish to express my protein so that it will be secreted into the periplasm. One issue is, no one in the lab actually attempted it and there are many info available but not a specific protocol (that I saw) with bacterial strains, expression vectors, helper stuff (e.g. pTUM4). I would also like to hear about your experience with periplasmic expression and what do you use and what you saw.


#2 protolder



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Posted 22 August 2011 - 12:05 AM

Hola, I used long time ago periplasmic vectors with ompA1signal peptide, piM-omp series of Inouye and express some litlle molecules ones with succes and some fails. I remember that the first GH was produced by Pharmacia with such systems. Nowadays there are some periplasmic vectors in the pET series (12, 20,22 25, 26 27) and more recently pET 39 and 40 with more posibilities of expression features for further purifications. Advantages : if it runs ok, you will get clean preparations. To isolate periplasmic proteins bacteria are plasmolized by osmotic shock (changing 20% sucrose swolen pellet culture to water giving the periplasmic fraction and espheroplasts. The induction has to be light , (inducer and temperature) because the processing machinery is the normal dotation of bacteria, so strong induction saturate processing machinery . The system always give a periplasmic fraction with processed protein and a cytoplasmic protein with non processed protein. I hope this helps to you. Buena suerte

#3 pDNA



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Posted 22 August 2011 - 12:59 PM

the pET system in combination with periplasmic secretion is most of the time far to strong and overburdens your system leading to slow/no growth and cell death. It is advisable to go for a weaker expression system like the tac system ...Sigma sells vectors of the FLAG series that have a tac promoter and a ompA signal sequence. They also have a protocol for osmotic shock for isolation. You can have a look [url="http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/flsbul.Par.0001.File.tmp/flsbul.pdf"]here[/url]!

Good luck!

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