I'm going to express recombinant spidroin from a pET-22b(+) plasmid, the protein MW is around 51 kDa and I want to study where the protein is expressed (extracellular, intracellular soluble/insoluble). I'm planning to concentrate the spidroin with ultrafiltration device, any advice on this? what parameter should be considered before doing this? Any input is appreciated, thank you in advance.
Hello Virania, is your goal is to purify the protein, determine where in the cell it is located, or both? Depending on the ultimate goal the recommendations will vary. Could you provide more information as to the organism and if this protein has been studied previously and if it is known to be insoluble? Thanks!
Hi, Jamison! My goal is both as the protein will be confirmed for western blot. Previously, the protein was expressed in E. coli BL21 (DE3) (same as what I do in this research) and known to be found in the insoluble fraction. This study can be accessed at https://pubmed.ncbi.nlm.nih.gov/32962298/. I'm planning to purify the protein with Ni-NTA chromatography, any advice?
Virania, it seems that Jia, Wen, and Ming (2020) obtained high yeilds of soluble spidroins from insoluble pellets without the use of metal-affinity chromatography. Is there a reason why you are not just using their method for purification? If you wish to know where the protein is being expressed, you could use eGFP (whole or split) and cell microscopy. If you do not want to purify from insoluble pellets you could explore solubility-enhancing tags (e.g., SUMO, MBP, eGFP, etc.), but, again, not sure if it is worth fixing a wheel that is not broken. I am sure there are other articles describing spidroin purification. I would recommend reviewing the literature on spidroin purification and see how folks have purified the protein. An answer that you’re looking for may already exist. Good luck!
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