Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences.
[EN] A copy of the bovine chymosin gene (chy) witha codon usage optimized for its expression in Aspergillusawamori was constructed starting from synthetic oligo-nucleotides. To study the ability of this filamentous fun-gus to secrete bovine prochymosin, two plasmids wereconstructed in which the tran...
| Autores: | , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2003 |
| País: | España |
| Institución: | Universidad de León |
| Repositorio: | BULERIA. Repositorio Institucional de la Universidad de León |
| OAI Identifier: | oai:buleria.unileon.es:10612/21809 |
| Acceso en línea: | http://hdl.handle.net/10612/21809 https://doi.org/10.1002/bit.10666 |
| Access Level: | acceso abierto |
| Palabra clave: | Biotecnología Filamentous fungi Synthetic chymosin gene Heterologous protein overexpression pH-regulated promoters 2414 Microbiología 2414.06 Hongos 2415.01 Biología Molecular de Microorganismos 3302.03 Microbiología Industrial |
| Sumario: | [EN] A copy of the bovine chymosin gene (chy) witha codon usage optimized for its expression in Aspergillusawamori was constructed starting from synthetic oligo-nucleotides. To study the ability of this filamentous fun-gus to secrete bovine prochymosin, two plasmids wereconstructed in which the transcriptional, translational,and secretory control regions of the A. nidulans gpdAgene and pepB genes were coupled to either preprochy-mosin or prochymosin genes. Secretion of a protein en-zymatically and immunologically indistinguishable frombovine chymosin was achieved in A. awamori transfor-mants with each of these constructions. In all cases,the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having amolecular weight of 35.6 kDa. Immunological assays in-dicated that most of the chymosin was secreted to theextracellular medium. Hybridization analysis of genomicDNA from chymosin transformants showed chromosom-al integration of prochymosin sequences and, in sometransformants, multiple copies of the expression cas-settes were observed. Expression from the gpdA pro-moter was constitutive, whereas expression from thepepB promoter was strongly influenced by pH. A veryhigh expression from the pepB promoter was observedduring the growth phase. The A. awamori pepB geneterminator was more favorable for chymosin productionthan the S. cerevisiae CYC1 terminator. |
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