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Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: Monitoring protein expression and solubility
Author(s) -
Cha Hyung Joon,
Wu ChiFang,
Valdes James J.,
Rao Govind,
Bentley William E.
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000305)67:5<565::aid-bit7>3.0.co;2-p
Subject(s) - green fluorescent protein , fusion protein , microbiology and biotechnology , chemistry , fluorescence , enteropeptidase , recombinant dna , biochemistry , expression vector , biology , plasmid , dna , gene , physics , quantum mechanics
We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His) 6 ‐GFP‐EK‐X, where X represents chloramphenicol acetyl‐transferase (CAT), human interleukin‐2 (hIL‐2), and organophosphorous hydrolase (OPH), respectively, (His) 6 represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein‐of‐interest from the fusion. The CAT and OPH fusion products (∼63 kDa GFP/CAT and ∼70 kDa GFP/OPH) were expressed at 4.85 μg/mL (19.9 μg/mg‐total protein) and 1.42 μg/mL (4.2 μg/mg‐total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL‐2 fusion (∼52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins‐of‐interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 565–574, 2000.