VECTOR FOR EXPRESSION OF HETEROLOGOUS PROTEIN AND METHODS FOR EXTRACTING RECOMBINANT PROTEIN AND FOR PURIFYING ISOLATED RECOMBINANT INSULIN

摘要

The present invention relates to a vector for expression of a heterologousprotein by a Gram negative bacteria, wherein the vectorincludes a nucleic acid such as DNA encoding the following: an origin ofreplication region; optionally and preferably a selection marker; apromoter; an initiation region such as translation initiation region and/or aribosome binding site, at least one restriction site for insertion ofheterologous nucleic acid, e.g. DNA, encoding the heterologous protein, and atranscription terminator. The inventive vector may containDNA encoding the heterologous protein, e.g., pro-insulin such as pro-insulinwith a His tag. Additionally, the invention provides a methodfor extracting a recombinant protein from within a recombinant Gram negativebacteria having a cell membrane, without lysing the bacteria,as well as a method for purifying an isolated recombinant human insulin,wherein the isolated recombinant human pro-insulin is subjectedto sulfitolysis, Ni-chelation chromatography, renaturation, limitedproteolysis and chromatography separation to provide purified, isolated,recombinant human insulin.