A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli.
The functional analysis of individual proteins or of multiprotein complexes - since the completion of several genome sequencing projects - is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.
Department Bio Pilot Plant, Leibniz-Institute for Natural Product Research, Infection Biology-Hans-Knöll-Institute, Jena, Germany.
Mario
M
Kraft
Uwe
U
Knüpfer
Rolf
R
Wenderoth
André
A
Kacholdt
Patricia
P
Pietschmann
Björn
B
Hock
Uwe
U
Horn
D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019897:Periplasm / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; C061682:DegP protease; D012697:Serine Endopeptidases