Expression of the human soluble low density lipoprotein receptor in methylotropic yeast.
- Author:
Jian HU
1
;
Yuan LI
Author Information
1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Binding Sites;
Electroporation;
methods;
Gene Expression;
Genetic Vectors;
Humans;
Ligands;
Pichia;
genetics;
Protein Structure, Tertiary;
genetics;
physiology;
Receptors, LDL;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
Reverse Transcriptase Polymerase Chain Reaction;
instrumentation
- From:
Chinese Journal of Biotechnology
2002;18(1):40-44
- CountryChina
- Language:Chinese
-
Abstract:
To obtain the expression of human low density lipoprotein receptor ligand binding domain in methylotropic yeast, firstly the DNA fragment encoding for human low density lipoprotein receptor ligand binding domain was amplified by RT-PCR with human hepatoma Bel-7402 total RNA as template. The nucleotide sequencing analysis indicated that the sequence of the cloned DNA fragment was as same as the reported human LDLR cDNA sequence. Then the expression vector pPIC9K-sLDLr was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sLDLR was identified by SDS-PAGE, Western blot and Ligand binding blot in supernatant of GS115/pPIC9K-sLDLr. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sLDLR was about 36 kD. And the ligand binding blot analysis indicated the expressed sLDLR has the biological activity. The sLDLR, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS115).
