Engineering green fluorescent protein as a dual functional tag.
A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.
Paramban
Rosanto I
RI
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Bugos
Robert C
RC
Su
Wei Wen
WW
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes; D005818:Genetic Engineering / Q000379:methods*; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D016254:Mutagenesis, Insertional; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014026:Nicotiana / Q000033:anatomy & histology
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D058619:Maltose-Binding Proteins; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine