Multipass Membrane Protein Research Articles

Anti-LAN Antibodies: Clinical Implications and Case Presentation

Abstract Introduction/Objective The LAN system, originating from the 901 Series of High Incidence antigens, transitioned to a recognized System in 2012. The LAN antigen, situated on ABCB6, a multipass membrane protein associated with porphyrin transport, plays a crucial role in heme synthesis. Functionally, LAN acts as an ATP-binding cassette transporter on the red blood cell membrane. Comprising a single antigen, LAN exhibits elevated expression in various tissues including the heart, skeletal muscles, fetal liver, mitochondrial membrane, eye, and Golgi apparatus. Notably, LAN shows no specific association with any particular geographic or ethnic group. Resistant to Ficin/Papain, Trypsin, α-Chymotrypsin, and 0.2M DTT, LAN antibodies primarily consist of IgG and necessitate IAT for detection. While Anti-LAN has been implicated in Hemolytic Transfusion Reactions (HTRs), its involvement in Hemolytic Disease of the Fetus and Newborn (HDFN) remains infrequent. LAN-negative blood is exceedingly rare, often requiring Monocyte Monolayer Assay Testing (MMA) for clinical assessment. Methods/Case Report Case Presentation: An 81-year-old female with a medical history including seizure disorder, hypertension (HTN), hyperlipidemia (HLD), gastroesophageal reflux disease (GERD), small bowel obstruction, and high-grade de-differentiated metastatic retroperitoneal liposarcoma presented for preoperative evaluation. A type and screen ordered as part of outpatient preoperative assessments revealed a positive antibody screen. Further investigation traced the origin of the anti-LAN antibody to an external facility since 2017. Antibody identification indicated the reactivity of the patient’s plasma with all cells (1+ AHG in PEG), excluding the auto control. Utilizing rare frozen antisera anti-LAN, the patient was phenotyped for LAN, yielding a LAN-negative status. Subsequent testing with three rare frozen LAN-negative group O cells aimed to exclude clinically significant antibodies. Differential adsorption, employing a comprehensive baseline RBC phenotype record obtained from the external facility, ruled out anti-E, anti-K, and anti-Fyb, thereby confirming Anti-LAN specificity. Results (if a Case Study enter NA) NA Conclusion The exceptional rarity of LAN-negative blood underscores the high prevalence of reactivity to Anti-LAN antibodies, exceeding 99% in most populations. In select cases, autologous transfusion may emerge as a viable management strategy. Consequently, the patient’s surgical procedure has been deferred, with no immediate requirement for blood products.

The dual loss and gain of function of the FPN1 iron exporter results in the ferroportin disease phenotype

Heterozygous mutations in SLC40A1, encoding a multi-pass membrane protein of the major facilitator superfamily known as ferroportin 1 (FPN1), are responsible for two distinct hereditary iron-overload diseases: ferroportin disease, which is associated with reduced FPN1 activity (i.e., decrease in cellular iron export), and SLC40A1-related hemochromatosis, which is associated with abnormally high FPN1 activity (i.e., resistance to hepcidin). Here, we report three SLC40A1 missense variants with opposite functional consequences. In cultured cells, the p.Arg40Gln and p.Ser47Phe substitutions partially reduced the ability of FPN1 to export iron and also partially reduced its sensitivity to hepcidin. The p.Ala350Val substitution had more profound effects, resulting in low FPN1 iron egress and weak FPN1/hepcidin interaction. Structural analyses helped to differentiate the first two substitutions, which are predicted to cause local instabilities, and the third, which is thought to prevent critical rigid-body movements that are essential to the iron transport cycle. The phenotypic traits observed in a total of 12 affected individuals are highly suggestive of ferroportin disease. Our findings dismantle the classical dualism of FPN1 loss versus gain of function, highlight some specific and unexpected functions of FPN1 transmembrane helices in the molecular mechanism of iron export and its regulation by hepcidin, and extend the spectrum of rare genetic variants that may cause ferroportin disease.

Getting membrane proteins into shape

In this issue, Ji etal.1 show how a multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase.

Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.

The Mass Spectrometry Identification, Antimicrobial Genes Detection, and Proteomics Analysis of Stutzerimonas stutzeri Strain Was Isolated from Industrial Wastewater

A large amount of organic matter, heavy metals, and even antibiotics are present in industrial wastewater, aquaculture waters, and various types of sewage, along with abundant microorganisms. To date, only a few studies involving the resistance and proteomics of Stutzerimonas stutzeri in high-salt wastewater are available. Herein, a comprehensive assessment of a newly isolated Stutzerimonas stutzeri strain, which is present in high-salt wastewater, was performed using mass spectrometry, genetic identification, and biochemical analysis to characterize the genetic and biochemical properties. Growth experiments revealed that the Stutzerimonas stutzeri strain had a moderate growth rate in nutrient broth, and the bacterial count was not high. Further analysis highlighted an apparent susceptibility of this strain to most antibiotics but some resistance to chloramphenicol and minocycline. A resistance gene assay results showed that the gene gyrB was associated with antibiotic resistance in this Stutzerimonas stutzeri strain. Proteomic analysis revealed for the first time the co-existence of two drug-resistance-related proteins (Multidrug/solvent RND membrane fusion protein and MexE) in Stutzerimonas stutzeri. Moreover, Stutzerimonas stutzeri isolated from high-salt wastewater was subjected to drug resistance gene detection, and the total protein of Stutzerimonas stutzeri was detected by protein mass spectrometry analysis. The subcellular classification shows that the 50 proteins with the highest abundance are divided into cell inner membrane, cell outer membrane, cytoplasm, cytoplasmic side, membrane, multi-pass membrane protein, and peripheral membrane protein, among which the proportion of cytoplasmic components is the highest. Overall, this study’s findings provide a new perspective for further research on the characteristics of Stutzerimonas stutzeri in high-salt wastewater.

The genetic network underlying the evolution of pathogenicity in avian Escherichia coli.

Colibacillosis is a worldwide prevalent disease in poultry production linked to Escherichia coli strains that belong to the avian pathogenic E. coli (APEC) pathotype. While many virulence factors have been linked to APEC isolates, no single gene or set of genes has been found to be exclusively associated with the pathotype. Moreover, a comprehensive description of the biological processes linked to APEC pathogenicity is currently lacking. In this study, we compiled a dataset of 2015 high-quality avian E. coli genomes from pathogenic and commensal isolates, based on publications from 2000 to 2021. We then conducted a genome-wide association study (GWAS) and integrated candidate gene identification with available protein-protein interaction data to decipher the genetic network underlying the biological processes connected to APEC pathogenicity. Our GWAS identified variations in gene content for 13 genes and SNPs in 3 different genes associated with APEC isolates, suggesting both gene-level and SNP-level variations contribute to APEC pathogenicity. Integrating protein-protein interaction data, we found that 15 of these genes clustered in the same genetic network, suggesting the pathogenicity of APEC might be due to the interplay of different regulated pathways. We also found novel candidate genes including an uncharacterized multi-pass membrane protein (yciC) and the outer membrane porin (ompD) as linked to APEC isolates. Our findings suggest that convergent pathways related to nutrient uptake from host cells and defense from host immune system play a major role in APEC pathogenicity. In addition, the dataset curated in this study represents a comprehensive historical genomic collection of avian E. coli isolates and constitutes a valuable resource for their comparative genomics investigations.

Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope1-5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

Electrophoretic characterization of cellular and extracellular proteins from Selenastrum capricornutum cultures degrading benzo(a)pyrene and their identification by UPLC-ESI-TOF mass spectrometry

Selenastrum capricornutum efficiently degrades benzo(a)pyrene (BaP) but few proteins related to BaP degradation have been identified in this microalgae. So far, it has only been suggested that it could degrade BaP via the monooxygenase and/or dioxygenase pathways. To know more about this fact, in this work, cultures of S. capricornutum incubated with BaP were used to obtain the molecular weights (MWs) of proteins existing in its extra- and cellular extracts by electrophoresis and UPLC-ESI(+)-TOF MS analysis. The results of this proteomic approach indicated that BaP markedly induces the MWs: 6–20, 30, 45, and 65 kDa in cells; 6–20, 30.3, 38–45, and 55 kDa in liquid medium. So, these proteins could be related to BaP biodegradation. An identified protein with monooxygenase activity and rubredoxins (Rds) show to be related to BaP degradation: Rds could participate, together with the monooxygenase in the electron transfer during the formation of monohydroxylated-BaP metabolites. Rds may be also associated with a dioxygenase system that degrades BaP to form dihydrodiol-BaP metabolites. A multi-pass membrane protein was identified too, and it can regulate the transport of molecules like enzymes from inside the cell to the outside environment. At the same time, the presence of a dihydrolipoamide acetyltransferase validated the stress caused by the exposure to BaP. It is noteworthy that these findings provide valuable and original information on the characterization of the proteins of S. capricornutum cultures degrading BaP, whose enzymes have so far not been known. It is important to highlight that the functions of the identified proteins can help in understanding the metabolic and environmental behavior of this microalgae, and the extracts containing the degrading enzymes could be utilized in bioremediation applications.

Mechanism of an intramembrane chaperone for multipass membrane proteins.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a locationbehind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity11. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endoplasmic reticulum.

Substrate-driven assembly of a translocon for multipass membrane proteins

Most membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes docked at the translocon, a heterogeneous ensemble of transmembrane factors operating on the nascent chain1,2. How the translocon coordinates the actions of these factors to accommodate its different substrates is not well understood. Here we define the composition, function and assembly of a translocon specialized for multipass membrane protein biogenesis3. This ‘multipass translocon’ is distinguished by three components that selectively bind the ribosome–Sec61 complex during multipass protein synthesis: the GET- and EMC-like (GEL), protein associated with translocon (PAT) and back of Sec61 (BOS) complexes. Analysis of insertion intermediates reveals how features of the nascent chain trigger multipass translocon assembly. Reconstitution studies demonstrate a role for multipass translocon components in protein topogenesis, and cells lacking these components show reduced multipass protein stability. These results establish the mechanism by which nascent multipass proteins selectively recruit the multipass translocon to facilitate their biogenesis. More broadly, they define the ER translocon as a dynamic assembly whose subunit composition adjusts co-translationally to accommodate the biosynthetic needs of its diverse range of substrates.

The Biogenesis of Multipass Membrane Proteins.

Multipass membrane proteins contain two or more α-helical transmembrane domains (TMDs) that span the lipid bilayer. They are inserted cotranslationally into the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum membrane. The Sec61 complex (SecY complex in prokaryotes) provides a ribosome docking site, houses a channel across the membrane, and contains a lateral gate that opens toward the lipid bilayer. Model multipass proteins can be stitched into the membrane by iteratively using Sec61's lateral gate for TMD insertion and its central pore for translocation of flanking domains. Native multipass proteins, with their diverse TMDs and complex topologies, often also rely on members of the Oxa1 family of translocation factors, the PAT complex chaperone, and other poorly understood factors. Here, we discuss the mechanisms of TMD insertion, highlight the limitations of an iterative insertion model, and propose a new hypothesis for multipass membrane protein biogenesis based on recent findings.

Identification of CD73 as the Antigen of an Antigen-Unknown Monoclonal Antibody Established by Exosome Immunization, and Its Antibody–Drug Conjugate Exerts an Antitumor Effect on Glioblastoma Cell Lines

Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibodies that recognized cancer-specific antigens on the exosome. In this study, we performed immunoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody–drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC has potential to be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization, which may allow the antibody to advance to new antibody therapies.

The mechanisms of integral membrane protein biogenesis.

Roughly one quarter of all genes code for integral membrane proteins that are inserted into the plasma membrane of prokaryotes or the endoplasmic reticulum membrane of eukaryotes. Multiple pathways are used for the targeting and insertion of membrane proteins on the basis of their topological and biophysical characteristics. Multipass membrane proteins span the membrane multiple times and face the additional challenges of intramembrane folding. In many cases, integral membrane proteins require assembly with other proteins to form multi-subunit membrane protein complexes. Recent biochemical and structural analyses have provided considerable clarity regarding the molecular basis of membrane protein targeting and insertion, with tantalizing new insights into the poorly understood processes of multipass membrane protein biogenesis and multi-subunit protein complex assembly.

Potential stress tolerance roles of barley germins and GLPs

Germins and germin-like proteins (GLPs) known as germination markers are encoded by multigene families in several plant species, including barley. To date, functional analysis has revealed germins and GLPs are involved in diverse processes such as embryonic development and stress responses. The aim of this study was the analysis of barley germins and GLPs. In this study, 80 putative germins and GLPs have been identified in barley by using known 17 germins and GLP sequences. Analysis of germins and GLPs showed all germins and GLPs are distributed on all seven chromosomes that most of them spread through chromosome 3, 4 and 7 with 16, 18 and 12 proteins on each, respectively. The protein sizes varied between 185 and 335 amino acids, with an average length of 225 aa. Twelve conserved motifs were found. While germin motifs 1 and 3 were detected in all germins and GLPs, some motifs were found to be related to signalization. Interestingly, protein-protein interaction analysis demonstrated some GLPs are associated with RAB6-interacting golgin, oligopeptide transmembrane transporter activity, beta-glucuronidase activity, protein N-linked glycosylation, multi-pass membrane protein, and proteins containing zinc finger (Znf) domain and RING (really interesting new gene)-type zinc finger domains. Our findings suggest that barley germins and GLPs may have diverse functions that make them important candidates for crop improvement.

Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

Nef homodimers down-regulate SERINC5 by AP-2–mediated endocytosis to promote HIV-1 infectivity

SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2-dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5.

An ER translocon for multi-pass membrane protein biogenesis.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Abstract 527: Isolation of large and diverse monoclonal antibody panels against the multipass membrane protein targets Kv1.3, P2X7, and Claudin 18.2

Abstract Monoclonal antibodies (MAbs) are a well-established treatment approach in oncology and other diseases. Nevertheless, multipass membrane proteins are largely inaccessible as MAb targets due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. Integral Molecular's MPS Antibody Discovery platform specifically addresses each of these challenges. Using this platform, we have isolated large and diverse panels of functional (agonist/antagonist) MAbs against targets including Claudin 18.2 and the ion channels P2X7 and Kv1.3. A key feature that enables the success of the platform is the use of divergent species for immunization. Chickens are immunized with a combination of DNA and Lipoparticles (high-concentration membrane proteins) to obtain high-titer immune responses against the native membrane protein. Our optimized phage-display protocols allow the isolation of large panels of MAbs against diverse epitopes. The MPS Antibody Discovery platform offers the potential for discovering MAbs against difficult targets in cancer with a success rate over 95%. Citation Format: Lewis J. Stafford, Sharon Willis, Joseph Rucker, Benjamin Doranz, Ross Chambers. Isolation of large and diverse monoclonal antibody panels against the multipass membrane protein targets Kv1.3, P2X7, and Claudin 18.2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 527.

Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition.

The intestinal absorption of cholesterol is mediated by a multipass membrane protein, Niemann-Pick C1-Like 1 (NPC1L1), the molecular target of a cholesterol lowering therapy ezetimibe. While ezetimibe gained Food and Drug Administration approval in 2002, its mechanism of action has remained unclear. Here, we present two cryo-electron microscopy structures of NPC1L1, one in its apo form and the other complexed with ezetimibe. The apo form represents an open state in which the N-terminal domain (NTD) interacts loosely with the rest of NPC1L1, leaving the NTD central cavity accessible for cholesterol loading. The ezetimibe-bound form signifies a closed state in which the NTD rotates ~60°, creating a continuous tunnel enabling cholesterol movement into the plasma membrane. Ezetimibe blocks cholesterol transport by occluding the tunnel instead of competing with cholesterol binding. These findings provide insight into the molecular mechanisms of NPC1L1-mediated cholesterol transport and ezetimibe inhibition, paving the way for more effective therapeutic development.

Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.