Recombinant production, isotope labeling and purification of ENOD40B: a plant peptide hormone.
The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.
Department of Chemistry and Institute for Chemical Biology, Sejong University, Seoul 143-747, Korea. ykchae@sejong.ac.kr
Young Kee
YK
Chae
Marco
M
Tonneli
John L
JL
Markley
D003001:Cloning, Molecular; D004926:Escherichia coli; D018506:Gene Expression Regulation, Plant; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D036361:Peptide Hormones / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010944:Plants / Q000737:chemistry / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
C405430:ENOD40 RNA; D009587:Nitrogen Isotopes; D036361:Peptide Hormones; D062085:RNA, Long Noncoding; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin