Author/Editor     Piškur, J; Kielland-Brandt, MC
Title     Folding and secretion of Saccharomyces cerevisiae carboxypeptidase Y are influenced by fusion with short heterologous peptides
Type     članek
Source     Biotechnol Appl Biochem
Vol. and No.     Letnik 18, št. 3
Publication year     1993
Volume     str. 239-57
Language     eng
Abstract     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, and the plasmids were introduced into a yeast mutant that mis-sorts vacuolar proteins. Translation efficiency of mRNA from the expression plasmids encoding hybrid 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.
Descriptors     AMINO ACID SEQUENCE
BASE SEQUENCE
CALCITONIN
CARBOXYPEPTIDASES
IMMUNOBLOTTING
MOLECULAR SEQUENCE DATA
OLIGOPEPTIDES
PLASMIDS
PROTEIN FOLDING
RNA, MESSENGER
RECOMBINANT FUSION PROTEINS
SACCHAROMYCES CEREVISIAE
SPECTROMETRY, FLUORESCENCE
THYMOPENTIN