Patent ID: 7858339

Claim:
A process for recovering refractile particles containing a heterologous polypeptide from bacterial periplasm in which the polypeptide is insoluble comprising: (a) culturing bacterial cells, which cells comprise nucleic acid encoding phage lysozyme, nucleic acid encoding the heterologous polypeptide, a signal sequence for secretion of the heterologous polypeptide, and separate and different inducible promoters for each of the nucleic acid encoding the phage lysozyme and the nucleic acid encoding the heterologous polypeptide, whereby the heterologous polypeptide is secreted into the periplasm of the bacteria as an aggregate and the phage lysozyme accumulates in the cytoplasmic compartment, wherein expression of the nucleic acid encoding the phage lysozyme is induced by the addition of an inducer after about 50% or more of the heterologous polypeptide has accumulated; (b) disrupting the cells mechanically to release the phage lysozyme so as to release refractile particles from cellular matrix; and (c) recovering the released refractile particles from the periplasm, whereby chloroform is not used in any step of the process, and wherein the recovery step minimizes co-recovery of cellular debris with the released refractile particles.