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Is knocking out nagZ the answer to routine production of O‐GlcNAc‐modified proteins in E. coli? (LB143)
Author(s) -
Macnaughtan Megan
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb143
Subject(s) - escherichia coli , chemistry , substrate (aquarium) , biochemistry , in vivo , tyrosine , transferase , enzyme , biology , gene , genetics , ecology
The overall goal of this research is to develop a robust co‐expression system to produce large quantities of O‐beta‐N‐acetylglucosamine (O‐GlcNAc)‐modified proteins using E. coli. It has been shown by several groups that O‐GlcNAc transferase (OGT) can be expressed in E. coli and can modify co‐expressed substrate proteins in vivo. The O‐GlcNAc‐modified protein yields from these co‐expressions are typically low, on the order of nanograms to micrograms, even though E. coli is routinely used to produce milligrams of unmodified protein. We have discovered that an endogenous beta‐N‐acetylglucosaminidase (NagZ) sabotages O‐GlcNAc‐modified protein yields by cleaving O‐GlcNAc from modified proteins in vivo. A nagZ knockout strain of E. coli was made, and co‐expression of human OGT with two substrate proteins, human cAMP responsive element‐binding protein (CREB1) and Abelson tyrosine‐kinase 2 (ABL2), show a temporal production of O‐GlcNAc‐modified protein. The current results do not lead to a definitive conclusion, but to more questions about E. coli and OGT.