title: Intracellular stability of alpha fragments of beta-galactosidase: effects of amino-terminally fused polypeptides.
abstract: Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D000069816:SEC Translocation Channels; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C070291:NusG protein, E coli; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; D014157:Transcription Factors; D001616:beta-Galactosidase