Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin.
The human immunodeficiency virus-1 Tat protein can efficiently enter cells when added exogenously in tissue culture. Using the transactivation activity of Tat as a measure of intracellular delivery, we found that the addition of hydrophobic groups to Tat potentiated its uptake. Biotin was the most promising of the reagents tested and we characterized this effect in more detail. When coupled through a cysteine thiol, the addition of a single biotin to Tat increased activity by about six-fold. Increased activity was only seen with reducible biotin analogs, as modification with noncleavable analogs is known to block Tat transactivation activity. Biotin had no effect on Tat uptake when mixed with Tat without cross-linking. Recently, Tat was used as a carrier to direct the uptake of heterologous proteins into cells. We have used RNase as a model system for studying Tat-mediated uptake and found that biotin also increased the delivery of a Tat37-58-RNase conjugate. The increased uptake of Tat and Tat conjugates by addition of hydrophobic groups may significantly enhance the usefulness of Tat as a delivery vehicle, and the approach may be applicable to other systems.
Chen
L L
LL
Biogen Incorporated, Cambridge, Massachusetts 02142, USA.
Frankel
A D
AD
Harder
J L
JL
Fawell
S
S
Barsoum
J
J
Pepinsky
B
B
D000595:Amino Acid Sequence; D001692:Biological Transport; D001710:Biotin / Q000737:chemistry*; D016923:Cell Death; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D015696:Gene Products, tat / Q000302:isolation & purification / Q000378:metabolism*; D017930:Genes, Reporter; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010446:Peptide Fragments; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D001710:Biotin; D012260:Ribonucleases