<i>In vitro</i>
            display technologies reveal novel biopharmaceutics

Rothe, Achim; Hosse, Ralf J.; Power, Barbara E.

doi:10.1096/fj.05-5650rev

Cited by 103 publications

(58 citation statements)

References 95 publications

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“…Along with combinatorial library technology, a variety of screening systems, such as phage display, ribosome display, etc., have been constructed. [1][2][3][4][5] Although a large library around 10 12 can be prepared using phage and mRNA display systems, the number of all sequences of proteins using 20 amino acids at each position is too large to be covered. For example, the number of possible protein sequences resulting from 100 amino acid residues is 10 130 .…”

Section: Introductionmentioning

confidence: 99%

Selection and structural analysis of de novo proteins from an α3β3 genetic library

et al. 2009

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The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding a3b3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both a-helix and b-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three a-helices and three b-strands as intended by its design. Thus, it would appear that artificially generated a3b3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.

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Section: Introductionmentioning

confidence: 99%

Selection and structural analysis of de novo proteins from an α3β3 genetic library

et al. 2009

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“…However, it would be advantageous to expand the options that natural peptides offer for the tumor targeting (4) by the identification of novel, artificial peptides. The identification and characterization of novel peptides that bind to cell-surface receptors overexpressed on tumors can be achieved by the screening of phage-display libraries (5). However, the expectations for this technology have not been met for in vivo applications.…”

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confidence: 99%

Influence of Chelate Conjugation on a Newly Identified Tumor-Targeting Peptide

Mier¹,

Zitzmann²,

Krämer

et al. 2007

Journal of Nuclear Medicine

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The transfer of peptide sequences identified by screening of phage-displayed libraries to clinical application is often difficult. This study investigated whether coupling of a new peptide, FROP-1, to the chelator 1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA) resulted in structural restriction and, consequently, improved binding and stability. Methods: The peptide FROP-1 was coupled to the chelator DOTA and labeled with 111 In. The structural changes caused by the addition of the chelator were determined by circular dichroism. The properties of this modified peptide were investigated in in vitro binding assays and monitored for kinetics, competition, and internalization as well as serum stability. A cell-type binding profile was established and the in vivo biodistribution was evaluated in a nude mouse model. Results: When compared with the free peptide without chelator, FROPDOTA revealed different cellular uptake kinetics, reaching a maximum at 2 h in vitro. The cells completely accumulated the tracer, and competition experiments revealed that 99.4% (FRO82-2 cells), 98.6% (MCF-7 cells), or 99.3% (average for 3 primary head and neck tumor cell lines) of tracer accumulation could be suppressed, revealing the specificity of this process. The internalization kinetics determined in MCF-7 cells supported this finding: After an incubation time of 180 min, the major fraction of FROPDOTA was trapped intracellularly. Serum stability experiments revealed an increase in stability due to the chelator, with a half-life of 71 min. Circular dichroism measurements indicated a fixed a-helix structure of FROPDOTA representing a strong change in secondary structure. In competition binding experiments, the binding constant (K D ) to FRO82-2 cells was determined to be 494 nM. Despite this avid binding affinity, the binding kinetics were found to be too slow to induce an uptake in vivo before clearance. Consequently, the biodistribution revealed a rapid renal and hepatobiliary clearance, with blood levels dropping from 5.48 6 0.26 %ID/g (percentage injected dose per gram) 5 min after injection to 0.77 6 0.15 %ID/g at 135 min after injection. Conclusion: This study revealed that peptides that are identified by display techniques may be underrated. Careful alteration of their structure will permit going beyond the possibilities that the limited pool of naturally occurring peptides provide for tumor targeting.

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“…164 In addition to phage display, fully synthetic antibody libraries have been mined successfully with ribosome display. 165 Ribosome display is an in vitro display technology 166 that does not require transformation of prokaryotic or eukaryotic cells. [167][168][169] Ribosome display has been employed to both accomplish and guide the affinity maturation of human mAbs.…”

Section: Discussionmentioning

confidence: 99%