Post-translational Regulation of Expression and Conformation of an Immunoglobulin Domain in Yeast Surface Display

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47’s extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47’s five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expression levels might indicate and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing

Vortex length, vortex energy and fractal dimension of superfluid turbulence at very low temperature

By assuming a self-similar structure for Kelvin waves along vortex loops with successive smaller scale features, we model the fractal dimension of a superfluid vortex tangle in the zero temperature limit. Our model assumes that at each step the total energy of the vortices is conserved, but the total length can change. We obtain a relation between the fractal dimension and the exponent describing how the vortex energy per unit length changes with the length scale. This relation does not depend on the specific model, and shows that if smaller length scales make a decreasing relative contribution to the energy per unit length of vortex lines, the fractal dimension will be higher than unity. Finally, for the sake of more concrete illustration, we relate the fractal dimension of the tangle to the scaling exponents of amplitude and wavelength of a cascade of Kelvin waves.Comment: 12 pages, 1 figur

Protein-Protein Fusion Catalyzed by Sortase A

Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein

Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening

Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-x[subscript L], Bcl-2, Bcl-w, Mcl-1, and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides, and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-x[subscript L] and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma, and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with K[subscript d] < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ and may guide the development of targeted Bfl-1 inhibitors.National Institute of General Medical Sciences (U.S.) (Award GM084181)National Institute of General Medical Sciences (U.S.) (Award P50-GM68762

Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs

An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

PurposeProstate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies.MethodsYeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors.ResultsThe A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 &gt; A11 &gt; C5 &gt; P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo.ConclusionOf the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study

Anisotropic atomic motions in structural analysis by low energy electron diffraction

The structure of the √3 × √3 R30° overlayer formed by CO molecules adsorbed on a Ru(0001) was analyzed by low energy electron diffraction. Anisotropic atomic motions under the influence of thermal excitation were taken into account by adopting the concept of split positions. Apart from considerable improvement in the structural refinement this technique provides information about dynamic processes. In particular, the molecular axis of the CO molecules was found to be tilted on the average by (12±3° at 150 K, which is attributed to excitation of the bending mode vibration (i.e., frustrated translation)

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential