Expression of a DNA fragment encoding the active domain of human TNF related apoptosis inducing ligand in pichia pastoris.
- Author:
Hong XU
1
;
Xin-Tian LAI
;
Kai YE
;
Hui-Wen MA
;
Kui HONG
Author Information
1. College of Pharmacy, Wuhan University, Wuhan 430072, China.
- Publication Type:Journal Article
- MeSH:
Apoptosis;
drug effects;
Cell Line, Tumor;
Electrophoresis, Polyacrylamide Gel;
Genetic Vectors;
genetics;
Humans;
Immunohistochemistry;
Pichia;
genetics;
metabolism;
Protein Structure, Tertiary;
genetics;
physiology;
TNF-Related Apoptosis-Inducing Ligand;
genetics;
metabolism;
pharmacology
- From:
Chinese Journal of Biotechnology
2003;19(2):163-167
- CountryChina
- Language:Chinese
-
Abstract:
Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands which has been reported in 1995. The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells. It is a type II transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis. A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector. Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease. After proved with DNA sequencing. the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with alpha-factor secreting signal peptide. The recombinant plasmid pPIC9K - TRAIL was transferred into P. pastoris cell by spheroplast transformation. The recombinant yeasts were identified by antibiotic G418 and Southern dot blot. The transformants (His+ Mut(s)) containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL. After inducing for 3 - 4 days, the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot. Two expressed protein bands whose appearant molecular weight were 19kD and 38kD, respectively, could be specifically recognized by polyclonal antibodies against human TRAIL. The 38kD protein might be a dimers of TRAIL in the culture supernatant. The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant. Biological activity assay, in vitro, indicated that the expressed protein could induce tumor cells apoptosis.
