Production of tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli.
An improved expression plasmid pET-insulin-like growth factor-2 (IGF2) was constructed and transferred into Escherichia coli BL21(DE3) for the expression of tilapia insulin-like growth factor-2. The recombinant insulin-like growth factor-2 was produced as inclusion bodies, and the recombinant insulin-like growth factor-2 content was as high as 10.3% of the total protein content. For production of recombinant insulin-like growth factor-2 in E. coli, pH-stat fed-batch cultures were used to achieve a high cell density culture. A cell concentration 183gl(-1) dry cell weight (DCW) was obtained after 30h cultivation and plasmid stability was maintained at high levels. Expression of insulin-like growth factor-2 was induced at three different cell concentrations, 50, 78.5, and 114.5gl(-1) dry cell weight. When cells were induced at a cell concentration of 114.5gl(-1) dry cell weight, the amount of insulin-like growth factor-2 produced was 9.69gl(-1) (11.3% of the total protein). Using a simple purification process including inclusion body isolation, denaturation, refolding and Ni-NTA affinity chromatography, 19.51mg of insulin-like growth factor-2 was obtained from a 22.5ml of culture, and the recovery yield was 20.5%. The biological activity of the purified IGF-2 was demonstrated as promoting the growth of four different cell lines by the colorimetric bioassay and the best growth stimulation ratio was obtained for the Balb/3T3 clone 31A cell line.
Hu
Shao-Yang
SY
Laboratory of Fermentation, Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, ROC.
Wu
Jen-Leih
JL
Huang
Jan-Hsiung
JH
D016475:3T3 Cells; D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D002999:Clone Cells; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D005347:Fibroblasts / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020539:Sequence Analysis, Protein; D017210:Tilapia; D013997:Time Factors; D015027:Zebrafish
D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II