Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli

Bedouelle, Hugues; Bassford, P J; Fowler, Audrée V.; Zabin, Irving; Beckwith, Jon; Hofnung, Maurice

doi:10.1038/285078a0

Cited by 265 publications

(115 citation statements)

References 25 publications

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“…MalE is exported into the bacterial periplasmic space by means of an N-terminal signal sequence which has a length of 26 residues and is cleaved during the export process. Mutations in the signal sequence can prevent MalE export and provoke its accumulation within the cytoplasm [5]. MalE is a protein binding maltose and maltodextrins which is necessary for the transport of these sugars across the bacterial envelope (reviewed in [I]).…”

Section: Production Inmentioning

confidence: 99%

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

Bedouelle

Duplay

1988

European Journal of Biochemistry

124

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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.

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Section: Production Inmentioning

confidence: 99%

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

Bedouelle

Duplay

1988

European Journal of Biochemistry

124

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“…Direct evidence for a role of these segments in the translocation process has been provided by genetic experiments showing that mutations affecting specific residues within the signals prevent passage of the polypeptides across the membrane (6) . In fact, DNA sequence analysis of mutant genes for altered polypeptides ofthe maltose binding protein of E. coli (9), which cannot be transferred to the periplasmic space, and for defective polypeptides of the lambda receptor protein (53), which do not reach the outer membrane and remain within the cytoplasm, has shown that all these mutations involved either substitutions in the signal sequence, from a single hydrophobic or uncharged amino acid to a charged one, or small deletions within the hydrophobic portion of the signals .…”

Section: Role Of Cotranslational Insertion Signals In Determining Thementioning

confidence: 99%

Mechanisms for the incorporation of proteins in membranes and organelles.

Sabatini¹,

Kreibich²,

Morimoto³

et al. 1982

The Journal of Cell Biology

869

281

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“…These signals share some common features (von Heijne, 1983): They are basic at the N-terminus, apolar in the middle, and have small, uncharged amino acid residues preceding the site of cleavage by the signal (or leader) peptidase. Genetic studies (Emr et al, 1978;Bassford & Beckwith, 1979;Bedouelle et al, 1980;Emr & Silhavy, 1980) have revealed that the integrity of the hydrophobic stretch of signal peptides is vital for protein transport across the membrane barrier.…”

mentioning

confidence: 99%

The chemistry and enzymology of the type I signal peptidases

et al. 1997

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The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membranebound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.

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Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli

Cited by 265 publications

References 25 publications

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

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

Mechanisms for the incorporation of proteins in membranes and organelles.

The chemistry and enzymology of the type I signal peptidases

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