Production in Escherichia coli and one‐step purification of bifunctional hybrid proteins which bind maltose

Abstract

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signalsequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.

The dimerization motif of the glycophorin A transmembrane segment in membranes: Importance of glycine residues

“…To test whether these proteins were correctly integrated into the inner membrane with the ToxR moiety facing the cytoplasm and the MalE domain exposed to the periplasmic space (Ntn -C,,,, Fig. I), they were tested for their ability to functionally complement a MalE deficient E. coli strain (PD28; Bedouelle & Duplay, 1988). This strain is unable to grow in minimal medium with maltose as the only carbon source.…”

Conformational and Functional Properties of an Undecapeptide Epitope Fused with the C-Terminal End of the Maltose Binding Protein

“…The E. coli strains HB2200 (malT recA1 hsdR17 hsdM + ) (Bedouelle & Duplay, 1988), HB2151malT (F + malT) (Martineau et al, 1990), RZ1032 (Hfr dut ung) (Kunkel et al, 1987), and PD28 (∆malE444 malT c 1 recA) (Duplay et al, 1984), plasmids pPD1, pPD127 (Duplay et al, 1984), pAB1 (Blondel & Bedouelle, 1990), and pTZ18R (Mead et al, 1986) and phage M13KO7 (Vieira & Messing, 1987) have been described.…”

“…The production of protein MalE from plasmid pPD1 and the productions of the MalE-P11 hybrid proteins from plasmid pPR1 and its mutant derivatives in strain PD28, the preparations of periplasmic extracts and shocked cells by osmotic shock, the purification of MalE and its derivatives by affinity chromatography on cross-linked amylose and elution with maltose (Bedouelle & Duplay, 1988), and the purification of the TrpB 2 subunit and the preparations of its apo and holo forms (Högberg-Raibaud & Goldberg, 1977) were performed as described previously. Monoclonal antibody mAb164 was produced by injection of hybridoma cells in the peritoneum of mice and purified by chromatography on a DEAEcellulose column as described (Djavadi-Ohaniance et al, 1984;Friguet et al, 1989a).…”

“…Proteins or protein fragments with very diverse structures and functions have been fused with the N-or C-terminal end of MalE. Both partners of these hybrid proteins, MalE and the passenger protein, remain functional in most cases (Bedouelle & Duplay, 1988;Blondel & Bedouelle, 1990, 1991Brégégère et al, 1994;Blondel et al, 1996;England et al, 1997). Antigenic peptides (e.g., viral epitopes) have been inserted in permissive sites within the sequence of MalE, i.e., sites such that the insertion does not perturb the localization, structure, and activity of MalE (Duplay et al, 1987).…”

“…MalE, a periplasmic protein of 370 residues, is widely used as a carrier protein (Bedouelle & Duplay, 1988;Guan et al, 1988). Its main advantage resides in its ease of purification, by affinity chromatography on cross-linked amylose, and the very mild conditions of elution, by competition with free maltose (Kellermann & Ferenci, 1982).…”