These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.
The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single-and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH 2 -terminal 115 residues of the Rad54 protein are necessary for this interaction. Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.The RAD52 epistasis group includes genes involved in homologous recombination in the yeast Saccharomyces cerevisiae. Mutations in these genes result in phenotypes that include an inability to repair double-stranded breaks, as well as defects in mitotic and meiotic recombination (1-4). The Rad51, Rad55, and Rad57 proteins show considerable homology to the Eschericia coli RecA protein, the paradigmatic prokaryotic strand transferase. Indeed, Rad51 protein has been shown to mediate strand exchange in vitro between homologous single-and double-stranded DNAs in the presence of replication protein A (RPA), 1 the yeast single-stranded DNA-binding protein (5). A number of results suggest that the Rad51 protein functions as part of a multiprotein complex in vivo. For example, the Rad51 and Rad52 proteins have been shown to bind one another both in vivo (6) and in vitro (7), and there is genetic evidence that Rfa1 is associated with the putative recombination complex (8, 9). Furthermore, experiments from the Berg (Stanford University School of Medicine) and Symington (Columbia College of Physicians and Surgeons) laboratories demonstrated that Rad51 protein binds to the Rad55 protein in vivo, which in turn interacts with Rad57 protein (10,11).In this report, we present both in vivo and in vitro evidence for a direct association between the Rad51 protein and the RAD54 gene product, another member of the RAD52 epistasis group (12, 13). The discovery of a Rad54-Rad51 protein interaction provides further support for the existence of a "protein machine" for mitotic recombination in yeast and raises the possibility that the Rad54 protein could play a direct role in strand exchange. EXPERIMENTAL PROCEDURESYeast Strains-The strain used for the two-hybrid experiments was CB14.1-9a (MAT␣ ade2 ura3 URA3::GAL1-lacZ his3 leu2 gal4Dgal80D trp1 lys2 LYS2::GAL1-HIS3). The wild-type strain employed for expression of the epitope-tagged proteins was W303-1A (MAT␣ ade2-1 can1-100 his3-11,15 leu2-3, 112 trp1-1 ura3-1). His6Rad51 protein was expressed in U687, a rad51⌬ mutant (MAT␣ ade2-1 can 1-100 his3- 11,15 leu2-3, 112 trp1-1 ura3-1 rad51::LEU2). U671 is a rad54⌬ mutant (MAT␣ ade2-1 can1-100, his3-11,15 leu2-3, 112 trp1-1 ura3-1 rad54::LEU2).Plasmids-The parent vectors for making GAL4 activation domain (AD) and DNA-bindi...
The mediator is an approximately 20 protein complex that is essential for the transcription of most genes in yeast. It is contacted by a number of gene-specific activators, but the details of these interactions are not well understood in most cases. Here, evidence is presented that the mediator component Gal11 represents at least one target of the Gal4 activation domain (AD). Deletion of Gal11 is shown to decrease the affinity of the Gal4 AD for the mediator, and direct binding of an N-terminal domain of Gal11 with the Gal4 AD is demonstrated. Quantitative studies, however, indicate that the K(D) of the 1:1 Gal4 AD--Gal11 complex is modest. Combined with in vivo data showing that Delta gal11 cells exhibit reduced, but still significant, Gal4-mediated gene expression, these results suggest that the dimeric activator might also contact another protein in the mediator in addition to Gal11.
Many methods have been developed to produce bispecific antibodies (BsAbs) for industrial application. However, huge challenges still remain in synthesizing whole length BsAbs, including their assembly, stability, immunogenicity, and pharmacodynamics. Here we present for first time a generic technology platform of generating bispecific IgG antibodies, “Bispecific Antibody by Protein Trans-splicing (BAPTS)”. Different from published methods, we assembled two parental antibody fragments in the hinge region by the protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/heavy chain mispairing. Utilizing this simple and efficient approach, there have been several BsAbs (CD3×HER2, CD3×EGFR, EGFR×HER2) synthesized to demonstrate its broad applicability. Correctly paired mAb arms were assembled to form BsAbs that were purified through protein A affinity chromatography to demonstrate industrial applicability at large scale. Further, the products were characterized through physical-biochemistry properties and biological activities to confirm expected quality of the products from “BAPTS”. More importantly, correct pairing was confirmed by mass spectrum. Proof-of-concept studies with CD3×HER2 BsAb (T-cell recruitment) demonstrated superior bioactivity compared with trastuzumab. The results of undetectable mispairing and high biological activity have indicated that this method has the potential to be utilized to manufacture BsAbs with high efficiency at industrial scale.
Background: Prolactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. Methods: In this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb. Results: PRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN-γ and TNF-α, which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb). Conclusions: Both in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.
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