[Establishment of suc2 signal sequence trap system].
The secretion of proteins depends on the signal peptide located to the N-terminal of the protein precursor. We established a genetic system in yeast to screen cDNA library for the signal peptide encoding sequences. To do it, we mutated genomic suc2 gene (encoding yeast invertase) of EGY48 by one-step gene disruption method, and got yeast cell lines without invertase expression (EGY48-delta suc). To get vector for library screening, we inserted suc2 gene encoding mature peptide of invertase downstream to yeast promoter P-ADH1, and multiple cloning sites for insertion of library is between suc2 and P-ADH1. EGY48-delta suc transformed with the vector can grow on the medium with glucose as carbon source, but not on the medium with raffinose. Signal peptide of suc2 and alpha chain of human interleukin-2 was fused in frame to suc2 gene, then the two resulting vectors were transformed into EGY48-delta suc, all the transformants can grow in the medium with either raffinose or glucose as carbon source. Hence, the system established here can discern cDNA encoding signal peptide from the one not encoding signal peptide.
Sun
Q
Q
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Wang
J S
JS
Li
R
R
Zhou
P
P
Huang
H Y
HY
Han
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D006026:Glycoside Hydrolases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D043324:beta-Fructofuranosidase
D021382:Protein Sorting Signals; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase