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Verfahren zur Synthese von loeslichen, rekombinanten Proteinen aus Bakterienzellen

High yield production of soluble recombinant protein in bacteria - with control of temperature during induction phase so that protein is present in lysate supernatant rather than inclusion bodies.
 
: Bernd, O.; Waschuetza, G.; Zakaria, H.; Otto, B.

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Frontpage ()

DE 1996-19604583 A: 19960208
DE 1996-19604583 A: 19960208
EP 1997-101265 A: 19970128
DE 19604583 C2: 19991118
EP 787804 B1: 20050323
C12N0015
C12P0021
C12N0015
German
Patent, Electronic Publication
Fraunhofer IGB ()

Abstract
Production of soluble recombinant protein (A) in bacteria that carry a plasmid containing the complete gene downstream of a promoter comprises: (a) growing the cells to optical density (OD) 0.4-0.6 at 600 nm; (b) cooling the culture to 20-26 deg. C over 15-40 min; (c) inducing synthesis by adding 10-55 mu M inducer (I); (d) carrying out the induction step at 20-26 deg. C for 3.5-6.5 hr; (e) separating the resulting cell pellet, in a buffer, into inclusion bodies (IB) and a protein-containing supernatant, and (f) isolating (A) exclusively from the supernatant. USE - The method is specified for production of interferon- gamma (IFN- gamma ) or its variants, including truncated and disulphide-bridged forms. ADVANTAGE - The method provides much increased yields compared with known methods, typically for IFN- gamma 50% against 10%, without the need for refolding of (A) to active form (refolding takes place in the bacterium). After induction, 20-60% of (A) is in soluble form and the rest in I B, compare 95% in IB in known methods where induction is at higher temperature. Soluble IFN- gamma is hardly antigenic, unlike material isolated from IB.

: http://publica.fraunhofer.de/documents/PX-39538.html