Transfection of proteins (transproteins)

TRANSFECTION OF PROTEINS (TRANSPROTEINS)

transfection ways (those marked in purple are useful also in vivo)

... every length :

RME of the protein. Hydrophilicity and protease resistance may be improved by PEGylation
cytoduction : the cytoplasm of a donor cell is trasferred to a recipient cell
pore-forming proteins (PFP) : streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105-106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all 3 toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology^ref
Diatos Peptide Vectors (DPV) (Vectocell^®), originating from human heparin binding proteins and/or anti-DNA antibodies , once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell^® peptides can drive intracellular delivery of molecules of varying molecular weight, including full-length active immunoglobulins, with efficiency often greater than that of the well characterized cell penetrating peptide Tat. The internalization of Vectocell^® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy dependent endocytosis process, involving cell membrane lipid rafts. This endocytosis occurs after binding of the cell penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated^ref.

... up to 50÷60 amino acids long :

chimaeric proteins from gene fusion with a cell-penetrating peptide (CPP) / protein transduction domain (PTD), ie a basic domain or a basic domain-containing protein. E.g. :

Tat 48-60 or Tat 47-57 (from HIV-1 )
Rev 34-50 (from HIV-1 )
VP22 (from HSV ) : doubtful efficacy !

intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22^ref

FGF
MAP KLAL
murine vascular endothelial cadherin 615-632 (pVEC)
galanin -mastoparans synthetic chimeras : mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. Mastoparan A (MP-A) has been isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado. See also mastoparan B (MP-B).

galparan : a synthetic chimera from amino acids 1-13 of galanin and mastoparan
transportan : a synthetic chimera from 12 N-terminal amino acids of galanin, a connecting Lys and the 14 C-terminal amino acids of mastoparan.

3^rd a-helix in homeodomain of homeotic proteins

pAntennapedia (pAntp) 43-58 / penetratin from Antennapedia
pls1 from Isl1

tranfection aims

protein therapy
chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely

CALI of natural protein has been limited by the need to microinject dye-labeled nonfunction-blocking mAb
CALI of transgenic proteins tagged with 1 or 2 small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye (ReAsH) requires no antibodies or microinjection, and inactivates each protein by 90% in <30 s of widefield illumination. ReAsH-mediated CALI acts largely via singlet oxygen

fluorophore-assisted light inactivation (FALI) affords the possibility of targeting a particular surface protein for destruction using the specificity of a mAb. Excitation of a fluorescein–antibody conjugate with blue light eventually (after 1 h) leads to the destruction of the antigen within close proximity^ref