Article

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/2010; 1798(12):2249-57. DOI: 10.1016/j.bbamem.2010.02.003
Source: PubMed

ABSTRACT Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.

1 Follower
 · 
110 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this work is to propose a mathematical model for the iron losses prediction in soft magnetic materials with any supply voltage. The starting point for the analysis is the knowledge of the iron losses with sinusoidal supply. The model is based on the separation of the losses contributions due to hysteresis, eddy current and excess losses with sinusoidal supply. Since any contribution depends on the supply voltage characteristics, it is possible to find a direct mathematical relationship between the iron losses contribution and the supply voltage characteristics. As a consequence, an iron losses prediction can be obtained with any supply voltage applied to the magnetic material. Finally, the experimental proofs of the proposed methods are reported
    Electric Machines and Drives Conference, 2001. IEMDC 2001. IEEE International; 02/2001
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The recent elucidation of molecular regulators of apoptosis and their roles in cellular oncogenesis has motivated the development of biomacromolecular anticancer therapeutics that can activate intracellular apoptotic signaling pathways. Pharmaceutical scientists have employed a variety of classes of biologics toward this goal, including antisense oligodeoxynucleotides, small interfering RNA, proteins, antibodies, and peptides. However, stability in the in vivo environment, tumor-specific biodistribution, cell internalization, and localization to the intracellular microenvironment where the targeted molecule is localized pose significant challenges that limit the ability to directly apply intracellular-acting, pro-apoptotic biologics for therapeutic use. Thus, approaches to improve the pharmaceutical properties of therapeutic biomacromolecules are of great significance and have included chemically modifying the bioactive molecule itself or formulation with auxiliary compounds. Recently, promising advances in delivery of pro-apoptotic biomacromolecular agents have been made using tools such as peptide "stapling", cell penetrating peptides, fusogenic peptides, liposomes, nanoparticles, smart polymers, and synergistic combinations of these components. This review will discuss the molecular mediators of cellular apoptosis, the respective mechanisms by which these mediators are dysregulated in cellular oncogenesis, the history and development of both nucleic-acid and amino-acid based drugs, and techniques to achieve intracellular delivery of these biologics. Finally, recent applications where pro-apoptotic functionality has been achieved through delivery of intracellular-acting biomacromolecular drugs will be highlighted.
    Current pharmaceutical design 02/2011; 17(3):293-319. DOI:10.2174/138161211795049642 · 3.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, since endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. This work aimed to study the use of aqueous two-phase micellar systems (ATPMS) from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv), which works as a biological indicator. The GFPuv extraction and LPS removal were evaluated in ATPMS, partition assays were carried out using pure GFPuv and cell lysate from Escherichia coli. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (KGFPuv > 1), and LPS removal into the micelle-rich phase (%REMLPS > 98%). GFPuv was partitioned preferentially into the micelle-poor phase due to excluded-volume interactions in the micelle-rich phase. Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.
Show more

Preview

Download
3 Downloads
Available from