Thanks in advance to everyone who answers my question. I want to raise a recombinant N-methylhydantoinase protein by pET expression vector system. The protein is a tetramer of two identical small subunits (70 kDa )and two identical large subunits (64 kDa). The relative total mass of protein would be 268 kDa. I have cloned, expressed, and purified the gene corresponding to the small subunit and large subunit separately. Now I want to merge (renature) both subunits of the recombinant protein. but I am little-bit confused with the buffer, temperature pH condition, and ratio of protein subunit for the proper renaturation. So kindly help me to fix this issue
Hi,
Co-express both large and small subunits in the same host cell may work if they are found in soluble fraction after cell lysis. In this way, you should use one subunit inserted in a pET vector and other subunit in a compatible vector as pACYC or both subunits cloned in the same vector as pETDuet. You mentioned above that you have already purified both subunits separately. In case of your purified subunits are stable in the same buffer you can mix together in a ratio 1:1 and make a dialysis overnight then run a gel exclusion chromatography to check if you have a complex formed by small and large subunits. Best wishes!
It depends on whether the subunits need each other for proper folding (e.g., Na/K-ATPase, where the beta-subunit serves - amongst others - as a chaperone for the catalytic alpha-subunit) or whether they can fold separately (e.g., haemoglobin). In the latter case, you can expect oligomer formation by just mixing the subunits, in the former you just may be able to achieve folding and oligomer formation by mixing the denatured subunits (urea, mercaptoethanol) and dialyse away the denaturants. Every protein is different...
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