Membrane docking of an aggregation-prone protein improves its solubilization

We used preS2-S′-β-galactosidase, a three domain fusion protein that aggregates extensively at 43 °C in the cytoplasm of Escherichia coli to search for multicopy suppressors of protein aggregation and inclusion bodies formation, and took advantage of the known differential solubility of preS2-S′-β-galactosidase at 37 and 43 °C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 °C. First, we demonstrate that the differential solubility of preS2-S′-β-galactosidase results in a lactose-positive phenotype at 37 °C as opposed to a lactose-negative phenotype at 43 °C. We searched for multicopy suppressors of preS2-S′-β-galactosidase aggregation at 43 °C by selecting pink lactose-positive colonies on a background of white lactose-negative colonies after transformation of bacteria with an E. coli gene bank. We found only two multicopy suppressors of preS2-S′-β-galactosidase aggregation at 43 °C, protein isoaspartate methyltransferase (PIMT) and the membrane components ChbBC of the N,N′-diacetylchitobiose phosphotransferase transporter.We have previously shown that PIMT overexpression reduces the level of isoaspartate in preS2-S′-β-galactosidase, increases its thermal stability and consequently helps in its solubilization at 43 °C (Kern et al., J. Bacteriol. 187, 1377–1383). In the present work, we show that ChbBC overexpression targets a fraction of preS2-S′-β-galactosidase to the membrane, and decreases its amount in inclusion bodies, which results in its decreased thermodenaturation and in a lactose-positive phenotype at 43 °C. Cross-linking experiments show that the inner membrane protein ChbC interacts with preS2-S′-β-galactosidase. Our results suggest that membrane docking of aggregation-prone proteins might be a useful method for their solubilization.