Title :
The Expression of Bovine Enterokinase Catalytic Subunit in Methylotropic Yeast Pichia pastoris
Author :
Fang, Zj ; Huang, H.
Author_Institution :
Dept. of Bioeng., Tianjin Univ., Tianjin, China
Abstract :
The objective of the study was to acquire the bovine enterokinase light chain (EKL) expressed in Pichia pastoris, which would be used in the cleavage and purification of fusion proteins. The fragment of EKL cDNA was obtained by RT-PCR from a sold bovine´s duodenal mucosa, then cloned into the pUCm-T cloning vector and sequenced. Compared with the sequence deposited in GenBank, the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pPIC9 expression plasmid. The recombinant vector pPIC9-EKL was linearized and introduced into Pichia pastoris GS115 with the method of PEG1000. The recombinant EKL was secreted in fermentation supernatant with molecular weight of 36 KDa. The yield of EKL was 32.8% of total supernatant protein. After separation and purification by STI resin affinity chromatography, the production of rEKL was about 10.9 ug/mL fermented solution, which had a specific activity of 2.88times107U/mg.
Keywords :
DNA; biochemistry; catalysis; chromatography; enzymes; fermentation; genetics; microorganisms; molecular biophysics; proteins; EKL cDNA; STI resin; bovine duodenal mucosa; bovine enterokinase catalytic expression; bovine enterokinase light chain; chromatography; cloned gene sequence; fermentation supernatant; fusion protein purification; methylotropic yeast Pichia pastoris; molecular weight; pUCm-T cloning vector; Biochemistry; Biological cells; Biomedical engineering; Bovine; Capacitive sensors; DNA; Fungi; Protein engineering; Purification; Sequences;
Conference_Titel :
Bioinformatics and Biomedical Engineering , 2009. ICBBE 2009. 3rd International Conference on
Conference_Location :
Beijing
Print_ISBN :
978-1-4244-2901-1
Electronic_ISBN :
978-1-4244-2902-8
DOI :
10.1109/ICBBE.2009.5163615
