scholarly journals Antibody Mediated Transduction of Therapeutic Proteins into Living Cells

2005 ◽  
Vol 5 ◽  
pp. 782-788 ◽  
Author(s):  
James E. Hansen ◽  
Richard H. Weisbart ◽  
Robert N. Nishimura
Keyword(s):  
Therapeutic Proteins ◽  
Disease Process ◽  
Antibody Fragment ◽  
Living Cells ◽  
Cell Penetrating Peptides ◽  
Biologically Active ◽  
Protein Transduction ◽  
Protein Therapy ◽  
Single Chain ◽  
Protein Transduction Domains

Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cellsin vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.

scholarly journals Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones

1998 ◽  
Vol 180 (11) ◽  
pp. 2830-2835 ◽  
Author(s):  
Sau-Ching Wu ◽  
Ruiqiong Ye ◽  
Xu-Chu Wu ◽  
Shi-Chung Ng ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Molecular Chaperones ◽  
Total Yield ◽  
Antibody Fragment ◽  
Biologically Active ◽  
Production Yield ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Enhanced Production ◽  
Secretory Production

ABSTRACT Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.

scholarly journals Efficient Protein Transduction Method Using Cationic Peptides and Lipids

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Kei Yamaguchi ◽  
Mari Inoue ◽  
Naoki Goshima
Keyword(s):  
Protein Delivery ◽  
Cationic Lipid ◽  
Living Cells ◽  
Protein Transduction ◽  
Cationic Peptides ◽  
Proton Sponge ◽  
Protein Transduction Domains ◽  
Cargo Proteins ◽  
Endosomal Vesicles ◽  
Novel Protein

Cationic peptides termed protein transduction domains (PTDs) have been shown to cross biological membranes efficiently. However, proteins transduced by PTDs become entrapped within the endosomal vesicles and are not delivered into organelles. We have developed a novel protein delivery system to enhance the proton sponge effect, which results in rupture of the endosomes, by using a mixture of Wr-T transporter peptide and a commercially available cationic lipid reagent. This peptide and cationic lipid reagent mixture efficiently delivers a variety of cargo proteins into living cells by releasing them from the endosomes.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors

2004 ◽  
Vol 78 (24) ◽  
pp. 13743-13754 ◽  
Author(s):  
Florian Kühnel ◽  
Bernd Schulte ◽  
Thomas Wirth ◽  
Norman Woller ◽  
Sonja Schäfers ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Protein Transduction ◽  
Virus Binding ◽  
Adenoviral Infection ◽  
Protein Transduction Domains ◽  
Oncolytic Therapy ◽  
Uptake Rates ◽  
Immunodeficiency Virus ◽  
Transactivator Of Transcription ◽  
Adenovirus Receptor

ABSTRACT Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs. CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CARex-PTD-mediated infection using CARex and inhibition experiments with heparin showed that binding of CARex-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CARex-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CARex-PTD to adenoviral particles. Consequently, the mechanism of CARex-PTD-mediated infection involves coating of the viral fiber knobs by CARex-PTD, rather than placement of CARex domains on cell surfaces. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CARex-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

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