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•Engineering of streptavidin for improved/tighter binding of the Strep-tag II peptide.•Discovery of several unexpected structural motifs in two of the binding site loops.•Elucidation ...of four representative crystal structures in complex with the Strep-tag II.•Structural analysis of the complex between Strep-Tactin XT and the Twin-Strep-tag.•The resulting avidity effect allows ultra-strong, yet reversible complexation of proteins.
The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide – instead of D-biotin recognized by the natural protein – to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.
Epithelial-to-mesenchymal transition (EMT) endows epithelia-derived cancer cells with properties of stem cells that govern cancer invasion and metastasis. Vimentin is one of the best studied EMT ...markers and recent reports indicate that vimentin interestingly translocated onto cell surface under various tumor conditions. We recently reported a cell surface vimentin (CSV) specific peptoid antagonist named JM3A. We now investigated the selective antagonist activity of the optimized homo-dimeric version of JM3A, JM3A-L2D on stem-like cancer cells or cancer stem cells (CSCs) over normal cells in non-small cell lung cancer (NSCLC). Homo-dimerization of JM3A provided the avidity effect and improved the biological activity compared to the monomeric version. We first optimized the central linker length of the dimer by designing seven JM3A derivatives with varying linker lengths/types and evaluated the anti-cancer activity using the standard MTS cell viability assay. The most optimized derivative contains a central lysine linker and two glycines, named JM3A-L2D, which displayed 100 nM vimentin binding affinity (K
) with an anti-cancer activity (IC
) of 6.7 µM on H1299 NSCLC cells. This is a 190-fold improvement in binding over the original JM3A. JM3A-L2D exhibited better potency on high vimentin-expressing NSCLC cells (H1299 and H460) compared to low vimentin-expressing NSCLC cells (H2122). No activity was observed on normal bronchial HBEC3-KT cells. The anti-CSC activity of JM3A-L2D was evaluated using the standard colony formation assay and JM3A-L2D disrupted the colony formation with IC
∼ 400 nM. In addition, JM3A-L2D inhibited cell migration activity at IC
∼ 2 µM, assessed via wound healing assay. The underlying mechanism of action seems to be the induction of apoptosis by JM3A-L2D on high-vimentin expressing H1229 and H460 NSCLC cells. Our optimized highly CSV selective peptoid has the potential to be developed as an anti-cancer drug candidate, especially considering the high serum stability and economical synthesis of peptoids.
The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can ...then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.
We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF).
The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.
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•Optimization of plectin targeted, cancer stem cell (CSC) specific peptoid PCS2D1.2.•PCS2D1.2 display highly specific cytotoxicity towards CSCs over non-CSCs.•PCS2D1.2 effectively ...blocked the in vitro colony formation and cell migration.•PCS2D1.2 reduced the in vivo tumor formation.•In both in vitro and in vivo studies, PCS2D1.2 reduced plectin-rich CSCs.
Cancers are highly heterogeneous and typically contain a small subset of drug-resisting cells called tumor initiating cells or cancer stem cells (CSCs). CSCs can self-renew, divide asymmetrically, and often cause tumor invasion and metastasis. Therefore, treatments specifically targeting CSCs are critical to improve patient survival. Recently, we identified a highly specific peptidomimetic (peptoid – PCS2) that selectively binds to the CSC subpopulation of lung cancer over the remaining cancer cells (non-CSCs). Subsequently, we identified plectin as the target of PCS2. Plectin is an intracellular structural protein, which is involved in tumor invasion and metastasis when it appears on cell surface. While PCS2 monomer did not display any anti-cancer activity, we designed a series of homo-dimeric versions of PCS2, and identified PCS2D1.2 optimized homo-dimer that displayed highly specific cytotoxicity towards CSCs over non-CSCs. PCS2D1.2 effectively blocked the in vitro colony formation and cell migration, hallmarks of CSCs. Furthermore, PCS2D1.2 reduced the in vivo tumor formation. In both in vitro and in vivo studies, PCS2D1.2 effectively reduced plectin expression and/or plectin-rich CSCs, but had no effect on non-CSCs. Therefore, PCS2D1.2 has the potential to be developed as a highly CSC specific drug candidate, which can be used in combination with current anti-cancer drugs.
Antibody is a very important protein in biotechnological and biomedical fields because of its high affinity and specificity to various antigens. Due to the rise of human antibody therapeutics, its ...cost-effective purification is an urgent issue for bio-industry. In this study, we made novel fusion proteins PAxPG with a flexible (DDAKK)n linker between the two Ig binding domains derived from Staphylococcus protein A and Streptococcus protein G. The fusion proteins bound human and mouse IgGs and their fragments with up to 58-times higher affinity and wider specificity than the parental binding domains. Interestingly, the optimal linker for human Fab fragment was n = 4, which was close to the modeled distance between the termini of domains bound to heavy chain, implying increased avidity as a possible mechanism. For binding to Fc, the longest n=6 linker gave the highest affinity, implying longer interchain distance between the two binding sites. The novel fusion protein with optimized interdomain linker length will be a useful tool for the purification and detection of various IgGs including mouse IgG1 that binds only weakly to natural protein A.
•A series of antibody binding proteins PAxPG is made from protein A and protein G.•Linkage of the domains by a flexible linker yielded higher affinity than the parents.•Optimization of the linker length resulted in higher affinity to hIgG Fab and Fc.•With its high affinity, PAxPG will be a useful probe to many Abs including mIgG1.
Abstract HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated ...by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects.
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