High Yield Expression and Purification of Human Endothelin-1
A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-α cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 μg/m...
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Published in | Protein expression and purification Vol. 5; no. 6; pp. 559 - 568 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.12.1994
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Subjects | |
Online Access | Get full text |
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Summary: | A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-α cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 μg/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with α-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 μg of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1046-5928 1096-0279 |
DOI: | 10.1006/prep.1994.1077 |