C - Chemistry – Metallurgy – 07 – K
Patent
C - Chemistry, Metallurgy
07
K
C07K 14/62 (2006.01) C07K 1/14 (2006.01) C07K 1/16 (2006.01) C12P 21/02 (2006.01) C12P 21/06 (2006.01) C12N 15/63 (2006.01) C12N 15/70 (2006.01)
Patent
CA 2687542
The present invention relates to a vector for expression of a heterologous protein by a Gram negative bacteria, wherein the vector includes a nucleic acid such as DNA encoding the following: an origin of replication region; optionally and preferably a selection marker; a promoter; an initiation region such as translation initiation region and/or a ribosome binding site, at least one restriction site for insertion of heterologous nucleic acid, e.g. DNA, encoding the heterologous protein, and a transcription terminator. The inventive vector may contain DNA encoding the heterologous protein, e.g., pro-insulin such as pro-insulin with a His tag. Additionally, the invention provides a method for extracting a recombinant protein from within a recombinant Gram negative bacteria 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 subjected to sulfitolysis, Ni-chelation chromatography, renaturation, limited proteolysis and chromatography separation to provide purified, isolated, recombinant human insulin.
Astolfi-Filho Spartaco
de Lima Beatriz Dolabela
de Sousa Heloisa Ribeiro Tunes
Thiemann Josef Ernst
Vilela Luciano
Biomm S.a.
Smart & Biggar
Universidade de Brasilia
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