Expression of honeybee prepromelittin as a fusion protein in Escherichia coli
| Autor: | Brian M. Austen, Denis Chapman, Jack A. Lucy, Ian M. Adcock, Mingyue He |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Signal peptide
Recombinant Fusion Proteins Molecular Sequence Data Gene Expression Biology medicine.disease_cause Inclusion bodies Gene expression Escherichia coli medicine Animals Amino Acid Sequence Protein Precursors Peptide sequence Signal peptidase Base Sequence DNA Bees beta-Galactosidase Melitten Molecular biology Fusion protein Secretory protein Biochemistry Plasmids Biotechnology |
| Zdroj: | Protein Expression and Purification. 2:363-371 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/1046-5928(91)90095-z |
| Popis: | Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful. |
| Databáze: | OpenAIRE |
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