I am trying to express a protein in K. lactis Protein Expression Kit (E1000S - NEB) in the way that the protein is secreted into the medium. I know that I can get some protein but it is not active. As the protein is secreted into the medium, I am not sure if doing expression for 24 h at 30°Cis a good idea. I am wondering if there are people here with some experience in this protein expression kit and, if so, what are the conditions for protein expression that worked well for you? Did you use ammonium sulphate precipitation to purify the protein from the medium or some other method?