Protein transduction: generation of full-length transducible proteins using the TAT system.
This unit describes the technology that allows an investigator to transduce full-length proteins by utilizing a minimal, eleven-amino acid, HIV-TAT transduction domain that can be fused to a protein of choice using the pTAT or pTAT-HA protein expression plasmids. Bacterial expression, followed by solubilization of protein aggregates with a denaturing agent, affords high yields of transducible fusion protein. The fusion protein, once added to the culture medium, can cross the cell membrane and then be degraded or refolded by the cellular machinery. Correct targeting and function of the fusion protein can be easily examined by fluorescent microscopy or immunohistochemistry. This strategy was established and improved to its current state by the purification and transduction of a multitude of fusion proteins. Because the pool of fusion proteins spans many different functions, the protocols cover a wide variety of commonly used protein isolation and characterization methods.
Becker-Hapak
Michelle
M
Washington University School of Medicine, Saint Louis, Missouri, USA.
Dowdy
Steven F
SF
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014161:Transduction, Genetic / Q000379:methods*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism
D011506:Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins