2 replies to this topic

[Andrea49]

Hey there,

I got a problem with my protein-expression.. I used the pRSET-system, host cells are E.coli BL21(DE3)pLysS, expected protein size app. 25kDa..  When I do the expression in small amounts (25ml LB+CAM+AMP in 250ml flask), it works quite well (increasing band 6h post induction with 1mM IPTG), although the total amount of the desired protein could be better... So I decided to upscale the protocol to 500ml LB (inoculated with 50ml of an over-night culture)in 2l-Flasks. Start-OD was nearly the same as in the small culture (app. 0,6), but I noticed, that the final OD 6h after IPTG-induction was much higher (small culture: app. 0,8-1,0, 500ml culture: over 2!). After lysis of cells (done by sonification) and Tricine-SDS-PAGE, i could no longer detect my band of interest... IPTG was freshly made, so this shouldn't be a matter.. Any guesses?

thanks in advance
Andrea

[protolder]

Andrea49, on 06 July 2011 - 08:44 AM, said:

Hey there,

I got a problem with my protein-expression.. I used the pRSET-system, host cells are E.coli BL21(DE3)pLysS, expected protein size app. 25kDa..  When I do the expression in small amounts (25ml LB+CAM+AMP in 250ml flask), it works quite well (increasing band 6h post induction with 1mM IPTG), although the total amount of the desired protein could be better... So I decided to upscale the protocol to 500ml LB (inoculated with 50ml of an over-night culture)in 2l-Flasks. Start-OD was nearly the same as in the small culture (app. 0,6), but I noticed, that the final OD 6h after IPTG-induction was much higher (small culture: app. 0,8-1,0, 500ml culture: over 2!). After lysis of cells (done by sonification) and Tricine-SDS-PAGE, i could no longer detect my band of interest... IPTG was freshly made, so this shouldn't be a matter.. Any guesses?

thanks in advance
Andrea

hola,if you see an increase of band in small scale, you have a quasi constitutive expression.so you have repress it before induction, because expression could kill expressing bacteria and survival of non expression ones. If I were you I would use lacIq subtype of this strain or before I will try expression with your strain in LB with 0.1g/l of glucose and 0.2g/l of lactose (natural inducer) and I will follow growth curve determining induction phase for growth or pH changes and analizying expression/time and I would finish at OD about 4. Buena suerte

[qzlabs]

You can try to cut down the induction time to 3 hours, and, when inoculating 2-liter new culture, spin down the cells and resuspend the cells in fresh medium. Using the cells in the fresh medium to inoculate the 2-liter culture. It should work. If you want a higher yield, you can try our new bioreactor (qizhonglabs.com) and use one of the auto-induction rich media. Please check out our website for more information at www.qizhonglabs.com.

Edited by qzlabs, 12 July 2011 - 12:53 PM.