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Secretion of human superoxide dismutase in Escherichia coli using the condensed single‐protein‐production system
Author(s) -
Mao Lili,
Stathopulos Peter B.,
Ikura Mitsuhiko,
Inouye Masayori
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.512
Subject(s) - periplasmic space , dsba , escherichia coli , superoxide dismutase , signal peptide , heteronuclear single quantum coherence spectroscopy , biochemistry , chemistry , heteronuclear molecule , peptide , biology , enzyme , recombinant dna , nuclear magnetic resonance spectroscopy , stereochemistry , gene
A secretion vector, pColdV for the Single‐Protein‐Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co‐expressed with MazF, an ACA‐specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ∼ 20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two SS bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope‐labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.