Protein Engineering Design and Selection - current issue

Using T7 phage display to select GFP-based binders

2008-06-13

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection.

Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability

2008-06-13

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug–drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.

Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Rice, J. J., Daugherty, P. S. — 2008-06-13

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.

Rapid and easy development of versatile tools to study protein/ligand interactions

2008-06-13

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the β-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid β-lactamases is achieved in the presence of β-lactams making further screening of correctly folded and secreted hybrid β-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the β-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the β-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

Miklossy, G., Tozser, J., Kadas, J., Ishima, R., Louis, J. M., Bagossi, P. — 2008-06-13

An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR_RE containing Asp25Arg and Gly49Glu mutations, and PR_RER containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. ¹H–¹⁵N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR_RE has a high tendency to aggregate, PR_RER exists mainly as a folded monomer at 25–35 µM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR_RE sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR_RER sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR_RER protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.

Expression, purification and characterization of BGERII: a novel pan-TGF{beta} inhibitor

2008-06-13

Transforming growth factor beta (TGFβ) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFβ isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFβ type II (RII) and betaglycan (BG) have been utilized as TGFβ inhibitors, their antagonistic potency against different TGFβ isoforms varies considerably because RII does not appreciably bind to TGFβ2 whereas BG binds weakly to TGFβ1 and TGFβ3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG_E) and the extracellular domain of RII. The fusion protein (named BG_ERII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFβ1 and TGFβ3 than a commercially available soluble RII and to TGFβ2 than a commercially available soluble BG. More significantly, whereas BG_E or RII alone showed no antagonistic activity towards TGFβ2, BG_ERII inhibited the signaling of both TGFβ1 and TGFβ2 in cell-based assays including TGFβ-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFβ-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG_ERII was found to have greater activity than other potent TGFβ inhibitors in blocking the signaling of TGFβ1 and TGFβ3. Thus, BG_ERII is a potent pan-TGFβ inhibitor in vitro and has potential for blocking TGFβ-induced pathogenesis in vivo.