Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning.
The efficient over-expression of several recombinant proteins in the same bacterial cell is usually prevented due to metabolic limitations. Nevertheless, the possibility to co-produce high amounts of the sub-units of a complex or to express a wide set of chaperones and foldases could be technologically very useful. We developed a system based on three vectors. Two are under IPTG regulation and enable the recombinant expression of six chaperones, the third one is arabinose-inducible and harbours the sequence for the target protein. In such a way the independent induction and the level of expression of both chaperones and target protein is possible. The data show that the expression leakage from pET vectors was prevented by the introduction of further plasmids in the cell and that the recombinant proteins compete for their expression. In fact, the high rate induction of one of them could switch off the accumulation of the other recombinant proteins. The first information was used to maximise the expression of toxic proteins while the cross-inhibition among recombinant proteins was exploited to modulate and optimise the target protein expression and to induce the chaperone-assisted in vivo re-folding of aggregated target protein.
de Marco
Ario
A
EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany. ario.demarco@embl-heidelberg.de
De Marco
Valeria
V
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001089:Arabinose / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D016547:Kinesins; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics*; D020559:Monomeric GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D009124:Muscle Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D014169:Transformation, Bacterial
D002135:Calcium-Binding Proteins; C121262:Gtr1 protein, S cerevisiae; D018832:Molecular Chaperones; D009124:Muscle Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C074152:kinesin-II; D007544:Isopropyl Thiogalactoside; D001089:Arabinose; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; D016547:Kinesins; D020559:Monomeric GTP-Binding Proteins