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KMID : 0545119980080060553
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Journal of Microbiology and Biotechnology
1998 Volume.8 No. 6 p.553 ~ p.559
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Expression of the EPO-like Domains of Human Thrombopoietin in Escherichia coli
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Koh, Yeo Wook
Koo,Tai Young/Ju, Sang Myoung/Kwon, Chang Hyuk/Chung, Joo Young/Park, Myung Hwan/Yang Jai Myung
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Abstract
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cDNA of human thrombopoietin (hTPO) amplified by polymerase chain reaction from a cDNA library of human fetal liver was cloned. EPO-like domains (hTPO_153 or hTPO_163) of hTPO (hTPO_332) were expressed in Escherichia coli using several kinds of expression systems, such as ompA secretion, thioredoxin fusion, and the P_L and T7 expression systems. To obtain hTPO_153 in soluble form, hTPO_153 cDNA was fused in-frame behind the gene encoding ompA signal sequence and thioredoxin protein. When fused with either of the genes, hTPO_153 was not expressed to the detectable level. However, a high level expression of the EPO-like domain of hTPO was obtained using the P_L and T7 expression system. hTPO_153 and hTPO_163 cDNA were subcloned into the pLex and pET-28a(+) vectors under the control of the inducible P_L and T7 promoter, respectively. Proteins expressed using pLex vector and pET-28a(+) detected in insoluble forms with an expression level of about 14% and 9% of total cellular proteins, respectively, and the level of expression was rapidly diminished in 2 h after the maximum level of expression was reached.
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