Hello,
I have cloned and expressed bacterial protein in E. coli system in the soluble fraction. However, on performing the activity assay, no activity with the substrate is seen. The standard assay procedure includes enzyme, substrate and buffer, and cold conditions have been maintained. I have cloned multiple genes and screened multiple proteins from the same strain, yet not one has given any activity. According to my understanding, bacterial genes are not pseudo in nature. Could someone suggest why this issue of inactive proteins is arising?
There can be many reasons for inactive proteins.
Check PI and pH of protein and choose buffer according.
Don't add Protese inhibitors... And wash ASAP from imidazole or other molecules if using for elution.
As you didn't give details how you measuring activity there can errors in that methods too
Hello Joyleen Maryann Fernandes
As Vijay Kumar suggested please give some details on how you are performing activity assay and also a method of protein purification. Also, use any positive control for your activity assay to confirm whether your purified protein is inactive or there is some other issue in your activity assay.
Thank you Vijay Kumar and Himanshu Gupta for your suggestions. The protein that is of our interest is a glycosyl hydrolase. The vector has a Histidine tag and hence Ni-NTA chromatography is being used for purification followed by desalting in order to remove imidazole molecules. The activity assay involves PNP-glucoside as substrate and the buffer is maintained at a pH of 6. The PI of our protein is 5. We have included a positive control i.e. Almond glucosidase which shows activity in the same system. Could you shed some light on this issue of inactivity using these details?
Joyleen Maryann Fernandes
I would suggest you change the pH of the elution buffer as some proteins lose their activity at some pH. Also, you can standardize the assay conditions (pH, time, temp) as different proteins behave differently.
Himanshu Gupta , I will implement this in my assay. Thank you!
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