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Protein Expression problems - (Feb/22/2012 )

Please suggest me

I am trying to clone 2 Kbp amylase gene into pET32a vector, after sucessful cloning into vector and transformation into BL21(DE3), it was checked by colony PCR and Restriction Digestion analysis., which confirm presence of my insert..The reading frame is also correct since I used a softwere to design primer which takes under considerstion of vector and insert also...all these are ok...but my problem start when I try to induced ....after induction using IPTG at 0.1-5mM conc at 0.6 OD for 6hrs I am getting in both the induced and non induced protein band pattern similar... on checking the activity of the transformed host cells and the lysate supernatant on a starch agar plate...it shows a clear zone of hydrolysis but I am not able to detect on SDS PAGE....so please suggest if any one could help me out these problem........I am not able to figure out the problem........

-jetu20-

Hi jetu20,

I can suggest a couple of possiblities but cannot be sure.

Firstly, you might have a low level of induction which means that you are getting enough protein to give enzymatic activity but not enough to see as a protein band. To see if this is the case, you could try a Western blot, which would make your protein stand out more, or you could vary your induction conditions, for example, inducing at 20oC, for 20 hours with 1 mM IPTG, in case more protein is produced.

Secondly, you may have leaky expression of your protein, meaning that it would be in the band pattern of the induced and non-induced samples. To investigate this possiblity you could do the agar plate test with the non-induced cells to see if they have activity too.

I hope this is some help

-Tehn-

Hola, in BL21 DE3 you can have a quasi constitutive expression without induction, because is a lacY+ strain. To detect your band you could compare the culture with your recombinant plasmid against the empty vector culture in BL21 too. Now a personal occurrence: In the field of gelatinases, zymograms detect the specific band after incubation of the run SDS gel (with gelatin added in the formula without reducer neither boil the sample) in a specific buffer overnight, and staining whith coomasie blue. Obviously as gelatin is a protein, the gel has a blue background and the gelatinase band has a clear zone by gelatin hydrolysis.I want to explain you that probably you can made a similar zymogram to amilases with any way to detect the starch hydrolysis as you see in agar plate. (any stain perhaps?). Think on it and have buena suerte

-protolder-

Hola again I have checked in internet and there are zymography methods for amylases in PAGE. one is in Mycologist (2005)19, 138-140 by Upadhyay, MK et al.

one more:


Arch. Biol. Sci., Belgrade, 62 (3), 575-583, 2010 DOI:10.2298/ABS1003575D
575
COMPARISON OF α-AMYLASE ISOFORMS FROM THE MIDGUT OF CERAMBYX CERDO L.
(COLEOPTERA: CERAMBYCIDAE) LARVAE DEVELOPED IN THE WILD AND ON AN
ARTIFICIAL DIET
BILJANA DOJNOV1*, N. LONČAR2, NATAŠA BOŽIĆ1, VERA. In this article free in internet, there are images of how the system runs. Buena suerte

-protolder-

Thanxs for your kind suggestion I would look into your suggestion and get back to you if I need some more suggestion....thanks once again.........

-jetu20-