Please use this identifier to cite or link to this item: http://nopr.niscair.res.in/handle/123456789/3418
Title: Overexpression of a recombinant -glutamyltranspeptidase from
Authors: Yao, Ya-Feng
Weng, Yih-Ming
Hu, Hui-Yu
Lin, Long-Liu
Keywords: Escherichia coli
-Glutamyltranspeptidase
Signal sequence
Overexpression
Nickel-chelate chromatography
Issue Date: 2006
Publisher: CSIR
Abstract: A truncated Escherichia coli Novablue -glutamyltranspeptidase (EcGGT) gene, lacking the first 48-bp coding sequence for part of the signal sequence, was amplified by polymerase chain reaction (PCR) and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20°C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 21 kDa respectively by SDS-PAGE, indicating the precursor EcGGT still undergoes the post-translational processing even in the truncation of signal sequence. His6-tagged EcGGT migrated relative to the molecular mass of approximately 120 kDa and its heterodimeric structure was confirmed by a native-PAGE gel.
Description: 345-350
URI: http://hdl.handle.net/123456789/3418
ISSN: 0301-1208
Appears in Collections:IJBB Vol.43(6) [December 2006]

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