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An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.

ABSTRACT: Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

SUBMITTER: Lan D

PROVIDER: S-EPMC7094418 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.

Lan Dongming D Huang Guangrui G Shao Hongwei H Zhang Lichun L Ma Lixin L Chen Shangwu S Xu Anlong A

Analytical biochemistry 20110427 2

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage ...[more]

PMID: 21600185

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Project description:Elastin-Like Polypeptides (ELP) are environmentally responsive protein polymers which are easy to engineer and biocompatible, making them ideal candidates as drug carriers. Our team has recently utilized ELPs fused to FKBP12 to carry Rapamycin (Rapa), a potent immunosuppressant. Through high affinity binding to Rapa, FKBP carriers can yield beneficial therapeutic effects and reduce the off-site toxicity of Rapa. Since ICAM-1 is significantly elevated at sites of inflammation in diverse diseases, we hypothesized that a molecularly targeted ELP carrier capable of binding ICAM-1 might have advantageous properties. Here we report on the design, characterization, pharmacokinetics, and biodistribution of a new ICAM-1-targeted ELP Rapa carrier (IBPAF) and its preliminary characterization in a murine model exhibiting elevated ICAM-1. Lacrimal glands (LG) of male NOD mice, a disease model recapitulating the autoimmune dacryoadenitis seen in Sjögren's Syndrome patients, were analyzed to confirm that ICAM-1 was significantly elevated in the LG relative to control male BALB/c mice (3.5-fold, p < 0.05, n = 6). In vitro studies showed that IBPAF had significantly higher binding to TNF-α-stimulated bEnd.3 cells which overexpress surface ICAM-1, relative to nontargeted control ELP (AF)(4.0-fold, p < 0.05). A pharmacokinetics study in male NOD mice showed no significant differences between AF and IBPAF for plasma half-life, clearance, and volume of distribution. However, both constructs maintained a higher level of Rapa in systemic circulation compared to free Rapa. Interestingly, in the male NOD mouse, the accumulation of IBPAF was significantly higher in homogenized LG extracts compared to AF at 2 h (8.6 ± 6.6% versus 1.3 ± 1.3%, respectively, n = 5, p < 0.05). This accumulation was transient with no differences detected at 8 or 24 h. This study describes the first ICAM-1 targeted protein-polymer carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in ICAM-1 overexpressing tissue in vivo, which may be useful for molecular targeting in diverse inflammatory diseases where ICAM-1 is elevated.

Dataset Information

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.

Publications

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.

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