High-level expression and purification of Tat-haFGF19-154

Abstract

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood-brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF(19-154) gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)-amplified coding sequence, the vector pTat-haFGF(19-154)-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF(19-154)-His was purified by a combination of Ni-NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF(19-154)-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED(50) was 3.931 x 10(-4) micromol/l. Tat-haFGF(19-154)-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.

The construction and characterization of two xylan‐degrading chimeric enzymes

“…Artificial chimeric proteins have been constructed for a variety of purposes including targeted delivery (Huang et al, 2008;Lu et al, 2006), improved stability (Eiben et al, 2007;Hua et al, 2004), novel specificity (Thulasiram et al, 2007;Zhang et al, 2007), and superior detection (Dan et al, 2008;Guan et al, 2007). Creation of artificial bifunctional, lignocellulosic hydrolases is a realistic and practical approach for the improvement of biomass conversion (Hong et al, 2006(Hong et al, , 2007Lu et al, 2006).…”

Tat-haFGF 14–154 Upregulates ADAM10 to Attenuate the Alzheimer Phenotype of APP/PS1 Mice through the PI3K-CREB-IRE1α/XBP1 Pathway

“…Intranasal delivery has proven to be a new and noninvasive strategy, which can circumvent the BBB and facilitate direct transport of the drugs to the brain through neuronal and extracellular pathways 44, 45. Our previous studies showed that intranasal administration of TAT-haFGF could improve cognition and reduce Aβ deposits more significantly in APP/PS1 mice compared with intravenous injection 18, 21, 22, 23. In an animal model of Parkinson’s disease induced by 6-OHDA, liposomes markedly assisted the delivery of basic fibroblast growth factor (bFGF) to the striatum and substantia nigra (SN), and they enhanced the neuroprotective effects of bFGF on dopaminergic neurons 46 .…”

“…Novel strategies were used to address this bottleneck problem, such as fusing the target protein with Tat-PTD 18 and utilizing intranasal delivery 19, 20. Our previous study showed that modified protein Tat-haFGF 14–154 could penetrate the BBB and was distributed in hippocampus and cortex via intravenous injection 18 . Following intranasal administration, the distribution of Tat-haFGF 14–154 was observed at 15 min in the brains of rats 21 .…”

A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

“…The fact that H3 Tat-3D8 was expressed in soluble form is itself noteworthy, because the soluble expression of Tat peptide-containing proteins (regardless of the position of the Tat peptide within the protein structure) has not yet been reported. Tat-fusion proteins have been purified from bacterial cell lysates only in insoluble form using urea-or guanidinium-denaturing protein purification methods, which require subsequent protein refolding [18][19][20][21]. However, we were unable to express soluble Tat-3D8 scFv proteins in which Tat 48-60 was fused to the N-terminus or grafted into VH CDR1 or CDR2 of 3D8 scFv using the same pIg20 expression vector (data not shown).…”