Construction and production of IGF feed yeast
Date Issued
2005-07-31
Date
2005-07-31
Author(s)
黃健雄
DOI
932313B002078
Abstract
The IGF-2 expression plasmid pGAP-IGF-2, constructed by the standard PCR method
using expression plasmid of E. coli pET-IGF-2 as the template, was linearized with
BglII and transformed to Candida utilis by electroporation. The IGF-2 transformant
was obtained by screening from the zeocin-contained YPD agar plates and molasses
agar plates sequentially. The transformant was characterized by Southern analysis.
After 48 h cultivation in molasses medium in shake flasks, 7.17 mg of IGF-2 per gram
of dried cell or 6.2 % of total soluble protein was achieved. IGF-2 could be purified
by Ni-NTA affinity chromatography. The N-terminal amino acid sequence of was
identical to that of the native IGF-2. Purified IGF-2 was further confirmed by
anti-mouse IGF-2 antibody and the molecular mass was 10717.9 Da by the mass
spectrum analysis, showing that there was no glycosylation. The bioactivity was
determined by measuring the extent of growth promotion of Balb/3T3 Clone 31A cell
line. A growth stimulation ratio of 74.18±10.73% was obtained in the presence of 120
nM of purified IGF-2. The bioactivity was 4.73±0.50 x 103 U mg-1. Ethanol of over
16 g l-1 in the medium would inhibit cell growth dramatically; however CSL could
enhance the cell growth. While fed-batch culture with constant feeding of glucose and
CSL in a 5-L fermentor, the cell concentration of 177.58 gDCW l-1 was obtained after
84 h cultivation and enthanol concentration in the medium was controlled under 10 g
l-1. The productivity was 2.114 g DCW l-1 h-1 and the yield based on glucose was 0.423 g g-1. The IGF-2 content was 5.2% of total soluble protein or 6.23 mg per gram
of dried cell. The bioactivity was 2.95 x 104 U gDCW-1.
Subjects
Insulin-like growth factor
electroporation
Candida utilis
transformant
bioactivity
Publisher
臺北市：國立臺灣大學生化科技學系
Type
report
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