Tat-mediated delivery of heterologous macromolecules into living cells

Barsoum, James; Moore, Claire; Seery, Joe; Daikh, Yasmin; Chen, Ling Ling; Corina, K; Moy, Pam; Brown, Rhonda; Shapiro, Reneé; Taylor, Fred; Androphy, Elliot J.; Pepinsky, Blake

doi:10.3233/rnn-1995-81203

Cited by 1 publication

(1 citation statement)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In fact, it was demonstrated that heterologous proteins chemically crosslinked to a domain of Tat protein from HIV were able to transduce into cells [13]: accordingly, this method become widely used because full-length proteins can be rapidly introduced into primary and immortalized cells. The fusion proteins can be directly added to cell culture [14,15] or injected in vivo into mice [16]. Protein transduction occurs in a concentration-dependent manner, requires at least one hour to achieve maximum intracellular concentrations with nearly equal intracellular concentrations among all cells in the transduced population and the uptake does not depend on the cell type used [17,18].…”

Section: Introductionmentioning

confidence: 99%

One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

2013

View full text Add to dashboard Cite

BackgroundHuman α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein.ResultsA reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range.ConclusionsTo further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

show abstract