Is your protein hydrophobic? Besides construct design, you need to consider other critical factors affecting expression. Some of the factors include:

1. Expression host strain

2. Media composition and use of the right antibiotics

3. Inoculum/Seed culture preparation

4. Pre-induction and post induction temperature

5. Concentration of IPTG

6. Post induction time

7. lysis buffer conditions if your protein is an intracellular protein

8. Your assay method for protein detection (SDS-PAGE vs Western blot)

9. other factors if any...........

Hi Johnson,

How do you check for protein expression? In case of protein targeted to extracellular space, especially when the level of expression is low, you will not see it directly in SDS-PAGE. I suggest TCA precipitation and/or WB.

Problems related to gene sequence:

1. If you did not optimize codons in your gene construct, the expression levels might not be satisfying. Try different strain for expression.

2. Unoptimized distance between RBS and first Met (recommended is 6-9 nt)

3. Changes in ORF

Other possible solutions:

1. IPTG quality might be questionable. I recommend confirming its activity.

2. Be sure that you do not have glucose in your medium, since it will repress expression in pET system.

3. Wrong condititions: if it is possible, you can check for expression after 4 hrs/8 hrs/16 hrs post induction & try different expression temperatures.

Good luck,

Marta

Dear Dr. Johnson Raja

The biological clock is very important for gene expression .

Best wishes

.

Dear Dr.Johnson Raja

Do you overcome to your problem? if yes, how?

Which bacterial strain you used?

You may try with other strain as well.