Published online Sep 15, 2000. doi: 10.3748/wjg.v6.iSuppl3.117
Revised: April 5, 2000
Accepted: May 10, 2000
Published online: September 15, 2000
AIM: To increase the production of recombinant des (1-3) IGF-I by increasing the copy number of gene carried on an expression vector, and to partially purify the expressed des (1-3) IGF-I, as well as compare its bio-activity with standard IGF-I.
METHODS: Second copy of des (1-3) IGF-I gene was inserted into pExSec1/IGF-I expression vector constructed by our previous work and carryed already one des (1-3) IGF-I gene, to form PExSec1/2 (IGF-I) expression plasmid, which carried two copies of tandem des (1-3) IGF-I gene. This plasmid was transformed into a protease deficient E. coli strain BL21 (DE3). The engineered bacteria was cultured and induced at low temperature. The expressed product was purified through ultra-filtration and gel-filtration. The bio-activity of partially purified protein was tested by MTT method and compared with standard IGF-I.
RESULTS: The amount of des (1-3) IGF-I expressed by pExSec1/2 (IGF-I) reached up to 19%-22% of the total soluble bacterial protein, which is about 7% higher than that of des (1-3) IGF-I expressed by pExSec1/IGF-I. The purity of recombinant des (1-3) IGF-I reached 49% and 82% respectively after the treatments by ultra filtration and gel-filtration. The result of MTT assay showed that the bio-activity of des (1-3) IGF-I after gel-filtration was about 77% of that of standard IGF-I at the same concentration.
CONCLUSION: The yield of recombinant des (1-3) IGF-I was increased about 7% by construction of expression plasmid with two copies of des (1-3) IGF-I gene, compared with only one copy of gene, preliminarily purified des (1-3) IGF-I showed relatively high biological activity, which was about 77% of that of standard IGF-I.