Α genetic fusion construct between the tetanus toxin C fragment and the lysosomal acid hydrolase β‐glucuronidase expresses a bifunctional protein with enhanced secretion and neuronal uptake

Jiang, Kanli; Watson, Deborah; Wolfe, John H.

doi:10.1111/j.1471-4159.2005.03133.x

Cited by 6 publications

(2 citation statements)

References 37 publications

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“…It is generally recognized that closer spatial distance between the fused proteins affects their folding, function, and expression. Flexible linkers are typically incorporated in cases where the fused domains require a certain degree of movement or interaction (Kim et al, 2000;Blazyk and Lippard, 2004;Jiang et al, 2005;Wang et al, 2007;Lu and Feng, 2008). Most commonly used flexible linker sequences primarily consist of Gly and Ser residues ("GS" linker) (Huston et al, 1988;Kavoosi et al, 2007;Hölsch and Weuster-Botz, 2010).…”

Section: Discussionmentioning

confidence: 99%

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Xu¹,

Ding²,

Cui³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS) 1 , L2: (GGGGS) 2 , and L3:(GGGGS) 3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins 19361Linker length affects HSA-fused ONC in P. pastoris ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 19360-19370 (2015) were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

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Section: Discussionmentioning

confidence: 99%

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Xu¹,

Ding²,

Cui³

et al. 2015

Genet. Mol. Res.

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“…2004), the receptor‐associated protein targeting the low‐density lipoprotein receptor (Prince et al. 2004), or the tetanus toxin C fragment (Jiang et al. 2005).…”

Section: Discussionmentioning

confidence: 99%

Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

Zhang

Dinh

Cronin

et al. 2008

Journal of Neurochemistry

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A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV‐1 Tat protein (Tat‐PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat‐PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C‐terminally with the Tat‐PTD. Compared with unmodified GALC, GALC bearing a Tat‐PTD, a myc epitope and 6 consecutive His residues [GALC‐TMH (Tat‐PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC‐TMH was found to be taken up by cells both via mannose‐6‐phosphate receptor (M6PR)‐mediated endocytosis as well as by M6PR‐independent mechanisms. GALC‐TMH displayed increased M6PR‐independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat‐PTD. Immunocytochemical analyses revealed that Tat‐modified GALC protein co‐localized in part with the lysosome‐associated membrane protein‐1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC‐TMH. Therefore, GALC‐TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.

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Presynaptic Neurotoxins with Enzymatic Activities

Rossetto

Montecucco

2008

Handbook of Experimental Pharmacology

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Toxins that alter neurotransmitter release from nerve terminals are of considerable scientific and clinical importance. Many advances were recently made in the understanding of their molecular mechanisms of action and use in human therapy. Here, we focus on presynaptic neurotoxins, which are very potent inhibitors of the neurotransmitter release because they are endowed with specific enzymatic activities: (1) clostridial neurotoxins with a metallo-proteolytic activity and (2) snake presynaptic neurotoxins with a phospholipase A2 activity.

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Α genetic fusion construct between the tetanus toxin C fragment and the lysosomal acid hydrolase β‐glucuronidase expresses a bifunctional protein with enhanced secretion and neuronal uptake

Cited by 6 publications

References 37 publications

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

Presynaptic Neurotoxins with Enzymatic Activities

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