دورية أكاديمية
Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris
| العنوان: | Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris |
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| المؤلفون: | WEISS, H. Markus, HAASE, Winfried, MICHEL, Hartmut, REILÄNDER, Helmut |
| المصدر: | Biochemical Journal ; volume 330, issue 3, page 1137-1147 ; ISSN 0264-6021 1470-8728 |
| بيانات النشر: | Portland Press Ltd. |
| سنة النشر: | 1998 |
| الوصف: | Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human β2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae α-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the β2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the α-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the β2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of β2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were ... |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| DOI: | 10.1042/bj3301137 |
| الإتاحة: | https://doi.org/10.1042/bj3301137Test https://portlandpress.com/biochemj/article-pdf/330/3/1137/632599/bj3301137.pdfTest |
| رقم الانضمام: | edsbas.B7B14584 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1042/bj3301137 |
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