RESUMO
A series of solvent-free elastin-like polypeptide liquid crystals and liquids are developed by electrostatic complexation of supercharged elastin-like polypeptides with surfactants. The smectic mesophases exhibit a high elasticity and the values can be easily tuned by varying the alkyl chain lengths of the surfactants or the lengths of the elastin-like polypeptides.
Assuntos
Elasticidade , Engenharia Genética , Cristais Líquidos/química , Peptídeos/química , Peptídeos/genética , Proteínas de Fluorescência Verde/genética , Modelos Moleculares , Conformação ProteicaRESUMO
Recombinant supercharged polypeptides (SUPs) with low cytotoxicity are developed and applied to rejuvenate the lubrication of naturally occurring salivary conditioning films (SCFs). SUPs with 72 positive charges adsorbed and rigidified the SCFs and recruited mucins to form a hydrated layer. These SCFs with SUPs have higher mechanical strength and sustain lubricating effect for longer duration compared with only SCFs.
Assuntos
Lubrificação , Peptídeos/química , Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Saliva/efeitos dos fármacos , Adsorção , Humanos , Saliva/metabolismoRESUMO
One of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.
Assuntos
Endocitose , Proteínas de Fluorescência Verde/metabolismo , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Morte Celular , Linhagem Celular Tumoral , Elastina/química , Elastina/metabolismo , Humanos , Espaço Intracelular/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismoRESUMO
Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv' variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv' molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv' molecules containing only one additional cysteine residue. In order to further improve the scFv' format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv' molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.