Xuefei Jin1, Dan Zhang2, Hongyan Li1, Ning Jin1, Tingting Liu3, Xiangbo Kong1. 1. Department of Urinary Surgery, China-Japan Union Hospital, Jilin University, Changchun, China. 2. Department of Endocrinology, China-Japan Union Hospital, Jilin University, Changchun 130033, China. 3. The Retired People in Army Residents of Jilin, Changchun, China.
Abstract
BACKGROUND: Matrix metalloproteinase 1 (MMP1) has been shown as a novel unique biomarker of bladder cancer in urine. MMP1 can only be detected using conventional and time-consuming methods, such as ELISA and Western. Refolded MMP1 has been achieved and used in probe screen for many years, while there is no clinical application for MMP1 detection until now. Soluble expression of MMP1 is necessary in urine detection. METHODS: cDNA of MMP1 has been isolated from human embryonic kidney 293(HEK293) cells. The catalytic domain of MMP1 is expressed as fusion protein with Escherichia coli thioredoxin (TrxA). The 30 kDa recombinant proteins were purified by Ni-chelating chromatography. The activity of soluble MMP1 was determined and compared with refolded MMP1 by zymography. RESULTS: Compared with refolded MMP1, TrxA can increase the solubility of MMP1. The soluble MMP1 has the same protein sequences with refolded MMP1 and increased 1.54-fold of gelatin-degradation activities than refolded MMP1. CONCLUSION: Successfully soluble expression of MMP1 has been achieved by fusion expression and will make progress in discovering specific molecular probes against MMP1.
BACKGROUND:Matrix metalloproteinase 1 (MMP1) has been shown as a novel unique biomarker of bladder cancer in urine. MMP1 can only be detected using conventional and time-consuming methods, such as ELISA and Western. Refolded MMP1 has been achieved and used in probe screen for many years, while there is no clinical application for MMP1 detection until now. Soluble expression of MMP1 is necessary in urine detection. METHODS: cDNA of MMP1 has been isolated from humanembryonic kidney 293(HEK293) cells. The catalytic domain of MMP1 is expressed as fusion protein with Escherichia coli thioredoxin (TrxA). The 30 kDa recombinant proteins were purified by Ni-chelating chromatography. The activity of soluble MMP1 was determined and compared with refolded MMP1 by zymography. RESULTS: Compared with refolded MMP1, TrxA can increase the solubility of MMP1. The soluble MMP1 has the same protein sequences with refolded MMP1 and increased 1.54-fold of gelatin-degradation activities than refolded MMP1. CONCLUSION: Successfully soluble expression of MMP1 has been achieved by fusion expression and will make progress in discovering specific molecular probes against MMP1.
Authors: Gregory P Botta; Mauricio J Reginato; Maximilian Reichert; Anil K Rustgi; Peter I Lelkes Journal: Mol Cancer Res Date: 2011-12-08 Impact factor: 5.852