Ubiquitin E3 Ligases

(DOCX) Click here for additional data file

(DOCX) Click here for additional data file.(22K, docx) Acknowledgments We gratefully acknowledge Yonghong Guan, Shalini Raphael, Thanh-Dung Nguyen and Hong Tong-Sevinc for excellent technical assistance. rule out potential interactions between SpA and the pentamerization domain (data not shown).(TIF) pone.0163113.s001.tif (18K) GUID:?454C251D-E7E6-41E4-89A6-A5D4E8CA4FEF S2 Fig: SpA binding by SPR of four llama VHH monomers (ICAM11-4, ICAM34-1, IGF1R-4 and IGF1R-5; dotted lines) and one llama VHH pentamer (AFAI; dotted line) along with their humanized counterparts (solid line). VHH monomers and pentamers (250 nM) were injected over immobilized SpA for 2 min and allowed to dissociate as described in methods.(TIF) pone.0163113.s002.tif (1.2M) GUID:?7CBACACF-5124-4976-8C15-D5B4B9B5FFA0 S3 Fig: Representative chromatogram Glimepiride overlay of purification of llama, humanized and SpA-engineered ICAM11-4 VHH. All three VHHs were produced in TG1 cells in 1L 2YT overnight cultures grown at 37C. VHHs were extracted from periplasmic space by osmotic shock and purified using a Glimepiride HiTrap Protein A HP column on an ?KTA FPLC protein purification system (GE Healthcare).(TIF) pone.0163113.s003.tif (163K) GUID:?DC426004-137C-4E0E-A3BF-27F28A9E7A10 S4 Fig: Binding of wild-type or SpA-engineered AFAI VHH pentamers at either 1 nM (A) or 50 nM (B) to immobilized CEACAM6 N-terminal domain by SPR. VHH pentamers were injected over immobilized CC2D1B CEACAM6 N-terminal domain for 3 min and allowed to dissociate as described in methods.(TIF) pone.0163113.s004.tif (300K) GUID:?B80074AB-64CD-4349-8A73-A401F8CEC413 S1 Table: FR sequences of SpA-binding and non-SpA-binding VHH monomers. (DOCX) pone.0163113.s005.docx (41K) GUID:?1AC6B0C7-08E3-487A-9649-F37785FBF045 S2 Table: FR sequences of SpA-binding and non-SpA-binding VHH pentamers. (DOCX) pone.0163113.s006.docx (26K) GUID:?641E0911-8B38-4A2F-9107-65F2040171E6 S3 Table: FR sequences of five non-SpA-binding VHHs and their humanized SpA-binding counterparts. (DOCX) pone.0163113.s007.docx (24K) GUID:?6FEE7BAE-FC81-448C-911A-637D337C2FEF S4 Table: Metrics for Illumina MiSeq NGS data used in this study. (DOCX) pone.0163113.s008.docx (20K) GUID:?DB05A66A-5027-446A-8085-12A00527404C S5 Table: FR and CDR sequences of a SpA-binding (Thr57) and non-SpA-binding (Ile57) dromedary VHH and effect of CDR1-CDR3 disulfide bridge on SpA binding. (DOCX) pone.0163113.s009.docx (22K) GUID:?C60D4B47-4D00-41FB-AF9A-B1BAF53304B6 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon non-SpA-binding VHHs. We show that SpA binding of VHHs is determined Glimepiride primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. Introduction Therapeutic antibodies (Abs) represent the fastest-growing class of biologic drugs, with expanding applications in cancer, chronic diseases and autoimmunity (reviewed in [1C3]). Currently licensed biologics are most commonly fully human or humanized monoclonal Abs (mAbs), with antigen-binding fragments such as Fab, F(ab)2 and scFv making up a smaller proportion of Glimepiride the market [4]. Next-generation Ab therapeutics will likely exploit the improved functional properties of molecularly engineered Abs, including bispecific Abs, Ab-drug conjugates and Fc-variant Abs [1,4,5]. VHH single-domain antibodies (VHHs), the variable domains of heavy-chain-only Abs produced naturally by camelid ungulates, may be useful in the design of next-generation biologics as a result of their small size, stability and modularity [6]. Full-length mAbs of all IgG subtypes can be easily purified by affinity chromatography, taking advantage of the high-affinity interactions between the IgG Fc and either staphyloccal protein.