FMDV-2A sequence and protein arrangement contribute to functionality of CYP2B1-reporter fusion protein.
Two optimized forms of green fluorescence proteins (GFP), enhanced GFP (EGFP) and humanized Renilla GFP (hrGFP), were used to track expression of cytochrome P450 2B1 (CYP2B1), an endoplasmic reticulum membrane-bound protein. In transiently expressing HEK293 cells we show that CYP2B1-GFP fusion proteins are stable and functional, whereas the vice-versa-arranged GFP-CYP2B1 fusions are not. The CYP2B1-hrGFP fusion protein is characterized by reduction in mean fluorescence intensity (MFI) to less than 20% of that of the hrGFP protein alone, accompanied by a 50% loss of CYP2B1 activity. Exchanging the linker for an alpha-helical peptide structure between CYP2B1 and hrGFP does not improve fusion protein activity. Insertion of a short linker (five amino acids) increases reporter protein fluorescence intensity twofold without improving CYP2B1 activity. Introduction of the foot and mouth disease virus 2A sequence providing cotranslational cleavage led to an unstable hrGFP-2A protein, whereas the corresponding EGFP-2A protein was stable and yielded an MFI superior to those of all other fusion constructs tested. CYP2B1 activity of the EGFP-2A-CYP2B1 protein was in the range of that of the unmodified CYP2B1. These data indicate that the protein arrangement EGFP-2A-CYP2B1 is superior to others, since it is most active and visible, which is essential for an effective tracking of the CYP2B1 enzyme.
Lengler
Johannes
J
Austrianova Biotechnology GmbH, University of Veterinary Medicine, A-1210 Vienna, Austria. lengler@austrianova.com
Holzmüller
Harry
H
Salmons
Brian
B
Günzburg
Walter H
WH
Renner
Matthias
M
D000818:Animals; D002460:Cell Line; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019362:Cytochrome P-450 CYP2B1