Gene construction, expression, and characterization of double-copy truncated form of human insulin-like growth factor I.
OBJECTIVE
To increase the production of recombinant truncated form of insulin-like growth factor I [des(1-3)IGF-I], purify the expressed product, and compare its bioactivity with that of standard IGF-I.


METHODS
The second copy of des(1-3)IGF-I gene was inserted into the previously constructed pExSec1/IGF-I to form a pExSec1/2(IGF-I) expression plasmid, then the plasmid was transformed into a protease-deficient E coli strain BL21(DE3). The engineered bacteria were cultured and induced by IPTG at 12 degrees C. The expressed product was purified through ultrafiltration and Sephadex G-50 gelfiltration. The bioactivity of the preliminarily 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 20 % of the total soluble bacterial protein, which was higher than the amount (12 %) expressed by a single copy of pExSec1/IGF-I gene. The purity of recombinant des(1-3)IGF-I reached 49 % and 82 % after ultrafiltration and gelfiltration. The bioactivity of des(1-3)IGF-I after gelfiltration was about 77 % of standard IGF-I at the same concentration.


CONCLUSIONS
The yield of recombinant des(1-3)IGF-I was increased about 8 % by construction of expression plasmid with two copies of des(1-3)IGF-I gene compared with only one copy of gene, and preliminarily purified des(1-3)IGF-I showed about 77 % bioactivity compared with standard IGF-I.
Sun
H Y
HY
Department of Life Science, Shanxi University, Taiyuan 030006, China.
Liu
X H
XH
Liang
S W
SW
D016475:3T3 Cells / Q000166:cytology; D000818:Animals; D001681:Biological Assay; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014462:Ultrafiltration
D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C065700:myotrophin