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Folding and secretion of Saccharomyces cerevisiae carboxypeptidase Y are influenced by fusion with short heterologous peptidesPiškur, Jure, 1960-2014 ; Kielland-Brandt, M. C.High levels of secreted yeast carboxypeptidase Y can be obtained in yeast by regulated overexpression under the control of the GAL1 promoter. Carboxypeptidase Y was investigated as a potential ... carrier for expression of heterologous oligopeptides. Coding sequences for two pentapeptides, Hepp (H-Asp-Ser-Asp-Pro-Arg-OH) and a thymopentin analogue (H-Arg-Pro-Asp-Val-Tyr-OH), and an analogue of salmon calcitonin, were inserted into, or added on to, the coding sequence for carboxypeptidase Y, andthe plasmids were introduced into a yeast mutant that mis-sorts vacuolar proteins. Translation efficiency of mRNA from the expression plasmids encodinghybrid carboxypeptidase Y was apparently not influenced by the insert.However, secretion of the hybrid proteins was lower than that of wild-type carboxypeptidase Y. A major fraction of the hybrid proteins accumulated intracellularly as a form characteristic for the endoplasmic reticulum. The results suggest that the inserted peptides influenced the secretion through the position and sequence effect on post-translational events. Furthermore, studies on folding properties indicated that the in vitro refolding capacity of the hybrid proteins was reduced. Thus, in the case of insertions, the transition from an unfolded chain to the correctly folded protein was likely to be disfavoured by misfolding. However, the tendency towards misfolding could be partially suppressed by changing the insertion site, and secretion was most effective in the cases of C-terminal fusions.Vir: Biotechnology and applied biochemistry. - ISSN 0885-4513 (Letn. 18, št. 3, 1993, str. 239-257)Vrsta gradiva - članek, sestavni del ; neleposlovje za odrasleLeto - 1993Jezik - angleškiCOBISS.SI-ID - 25493977
Avtor
Piškur, Jure, 1960-2014 |
Kielland-Brandt, M. C.
Teme
Amino Acid Sequence |
Base Sequence |
Calcitonin |
Chemistry |
Metabolism |
Carboxypeptidases |
Chemistry |
Genetics |
Secretion |
Immunoblotting |
Molecular Sequence Data |
Oligopeptides |
Biosynthesis |
Chemistry |
Plasmids |
Protein Folding |
RNA, Messenger |
Genetics |
Metabolism |
Recombinant Fusion Proteins |
Biosynthesis |
Chemistry |
Isolation & Purification |
Saccharomyces Cerevisiae |
Enzymology |
Spectrometry, Fluorescence |
Thymopentin |
Analogs & Derivatives |
RNA, prenašalna |
Saccharomyces cerevisiae |
Molekulsko zaporedje, podatki |
Rekombinantne, fuzijske beljakovine |
Karboksipeptidaze |
Imunoblot |
Baze, zaporedje |
Kalcitonin |
Plazmidi |
Beljakovine, gubanje |
Timopentin |
Aminokislinsko zaporedje |
Oligopeptidi |
Spektrometrija fluorescenčna
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