Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.
Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Ji-sun
JS
Kim
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Dong-Ki
DK
Choi
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Seong-wook
SW
Park
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Seung-Min
SM
Shin
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Jeomil
J
Bae
Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea.
Dong-Myung
DM
Kim
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea.
Tae Hyeon
TH
Yoo
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea. Electronic address: kimys@ajou.ac.kr.
Yong-Sung
YS
Kim
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D049452:Green Fluorescent Proteins
Cytosol localization; Cytosol-penetrating antibody; Endosomal escape; Split GFP; Streptavidin