pRSET expression system troubles

Vayuputra vayuputra369 at hotmail.com
Mon Aug 19 18:29:36 EST 2002


I've done some work with the pRSET vectors and had decent success.

One of my observations was that in small cultures (3-5 ml) I couldn't detect
the protein.  But when a large culture is grown (500 ml - 1 litre) I saw an
obvious overexpression.

OD values seem to be specific to individual fusion proteins - try various
conditions (induce the cells in increasing cell densities : 0.4, 0.6, 0.8
and 1.0 OD600 values).

Try 0.1mM, 0.5mM IPTG concentrations to induce the protein.

(These can only be done after following another suggestion to your query -
sequence the clones to verify if your protein is in frame with the tag.
Some clones get induced better than others.  Once I identified cell clones
expressing well I made glycerol stocks and have been using those cells
eversince).

Hope this helps.
Vayuputra





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