Expression, isolation, and characterization of a signal sequence-appended chimeric precursor protein.
This report describes the properties and the functional utility of an unprocessed precursor protein overproduced in Escherichia coli. The precursor protein is from a fusion between DNA sequences coding for the alkaline phosphatase signal sequence and the full-length of rat liver cytochrome b(5). The intact precursor protein accumulated in the membranes represented to over 5% of the total bacterial protein. A procedure involving disruption of the bacterial cells by sonication, isolation of the membranes by differential centrifugation, solubilization with a polar solvent, and ion-exchange chromatography provided milligram quantities of the undegraded precursor in a homogeneous and soluble form. The chimeric precursor protein displayed a characteristic b-type hemoprotein spectrum, identical to that of the native cytochrome b(5). The properties of the precursor protein have been examined by a range of biophysical and biochemical methods. Molecular modeling suggests an amphipathic structure in which a fully preserved soluble core of cytochrome b(5) is terminally bonded by hydrophobic interactions between the amino-terminal signal sequence and the carboxy-terminal membrane anchoring hemoprotein sequence. The precursor substrate was recognized and efficiently cleaved by signal peptidase.
Kaderbhai
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Institute of Biological Sciences, The University of Wales, Aberystwyth, Dyfed, United Kingdom.
Kaderbhai
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MA
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000096:biosynthesis*; D021382:Protein Sorting Signals / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000378:metabolism
D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; C026515:type I signal peptidase