IN-VIVO ASSEMBLY OF ACTIVE MALTOSE-BINDING PROTEIN FROM INDEPENDENTLYEXPORTED PROTEIN-FRAGMENTS

The maltose binding protein (MBP or MalE) of Escherichia coli is the p eriplasmic component of the transport system for malto-oligosaccharide s. It is synthesized in the cytoplasm with an N-terminal signal peptid e that is cleaved upon export. We examined whether active MBP could as semble into an active protein in bacteria, from N- and COOH-terminal c omplementary protein fragments encoded by distinct, engineered segment s of its structural gene. We found export and functional periplasmic a ssembly of MBP fragments, despite the complex polypeptide chain topolo gy of this protein, if two conditions were satisfied. First, each of t he two fragments must carry a signal peptide. Second, the boundaries b etween the two fragments must correspond to a permissive site within t he protein. Functional assembly of active MBP occurred in five cases w here these conditions were met: sites after residues 133, 161, 206, 28 5 and 303; but not in three other cases where the break junction corre sponded to a non-permissive site: after residues 31, 120 and 339. Thus , permissive sites which were initially characterized because they cou ld accept extensive genetic insertion/deletion modifications without l oss of most biological properties provide a means of defining compleme nting protein fragments. This observation opens a way to study genetic ally the relationships between protein export and folding into the per iplasm.