Hi,
I am trying to express Trypanothione synthetase a protein from Leishmania, cloned in pET28a vector and transformed in E.coli. BL21 (DE3). The size of the protein is 74kDa. Initially, I optimized the expression conditions which were as follows.
1. The cells were induced with 0.5mM IPTG in a 500ml primary culture (OD600= 0.6)
2. The induction temperature was 25 degree Celsius.
3. The incubation time was 16 hrs.
While repeating the same I am not getting the expression in either unpurified or purified condition.
For cell lysis, the buffer composition includes 50mM Tris-HCl pH8.0, 250mM NaCl, 0.2mM BME, 5% glycerol, and 1mM PMSF as the final working concentration. The cell lysis was done by sonication just after harvesting the cells at 10000rpm.
For purification, I am using the Ni-NTA column for purification of protein. I am using 50mM imidazole for washing and eluted with 200mM imidazole.
I tried the same procedure 3-4 times but unfortunately, I was unable to get the protein.
Please help with your valuable experience
There are some possibilities:
a) there is virus contamination in the procedure; incubator, baker or etc. you should check it out if you work with virus in the lab.
b) when you want to induce your culture; check the optical density (600 nm) of your culture, normally OD around 0.7~0.8 is good for induction bacterial expression (OD, IPTG concentration also should optimize; 1 mM IPTG normally get good result)
c)after sonicating your culture on ice, centrifuge at a higher speed for 30 minutes to remove cell debris (8000 rpm)
d) elute your protein from His_column with 500 mM imidazole.
As I understand your protein is an enzyme and if it's not thermostable you should do all the steps on ice and try to do purification and buffer exchange in one day!
These points come to my mind, hope it works!
Good luck~
Try re-transforming the cells with the plasmid. It may have been lost from the starter culture.
Hello
1. OD 0.7-0.9 is ideal for promoter induction.
2. Did you perform time-course study to determine the best induction time on both supernatant and pellet?
3. Try adding PMSF after sonicating of samples, incubate it with the samples for about 1hrs with gentle shaking at room temperature; thereafter perform centrifugation and do protein purification on suitable cell fraction (supernatant or pellet)
3. You could optimize imidazole concentration (0.5 -500 mM) by checking the protein concentration in eluted samples based on the Bradford assay.
Thank you everyone for your valuable suggestions. I noted down some important suggestions to follow. I hope the result will be positive.
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