High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size.

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.

Functional Expression, Purification and Identification of Interaction Partners of PACRG.

PACRG (Parkin co-regulated gene) shares a bi-directional promoter with the Parkinson's disease-associated gene Parkin, but the physiological roles of PACRG have not yet been fully elucidated. Recombinant expression methods are indispensable for protein structural and functional studies. In this study, the coding region of PACRG was cloned to a conventional vector pQE80L, as well as two cold-shock vectors pCold II and pCold-GST, respectively. The constructs were transformed into Escherichia coli (DE3), and the target proteins were overexpressed. The results showed that the cold-shock vectors are more suitable for PACRG expression. The soluble recombinant proteins were purified with Ni2+ chelating column, glutathione S-transferase (GST) affinity chromatography and gel filtration. His6 pull down assay and LC-MS/MS were carried out for identification of PACRG-binding proteins in HEK293T cell lysates, and a total number of 74 proteins were identified as potential interaction partners of PACRG. GO (Gene ontology) enrichment analysis (FunRich) of the 74 proteins revealed multiple molecular functions and biological processes. The highest proportion of the 74 proteins functioned as transcription regulator and transcription factor activity, suggesting that PACRG may play important roles in regulation of gene transcription.

Mechanistic Insights into the Role of Molecular Chaperones in Protein Misfolding Diseases: From Molecular Recognition to Amyloid Disassembly.

Age-dependent alterations in the proteostasis network are crucial in the progress of prevalent neurodegenerative diseases, such as Alzheimer's, Parkinson's, or amyotrophic lateral sclerosis, which are characterized by the presence of insoluble protein deposits in degenerating neurons. Because molecular chaperones deter misfolded protein aggregation, regulate functional phase separation, and even dissolve noxious aggregates, they are considered major sentinels impeding the molecular processes that lead to cell damage in the course of these diseases. Indeed, members of the chaperome, such as molecular chaperones and co-chaperones, are increasingly recognized as therapeutic targets for the development of treatments against degenerative proteinopathies. Chaperones must recognize diverse toxic clients of different orders (soluble proteins, biomolecular condensates, organized protein aggregates). It is therefore critical to understand the basis of the selective chaperone recognition to discern the mechanisms of action of chaperones in protein conformational diseases. This review aimed to define the selective interplay between chaperones and toxic client proteins and the basis for the protective role of these interactions. The presence and availability of chaperone recognition motifs in soluble proteins and in insoluble aggregates, both functional and pathogenic, are discussed. Finally, the formation of aberrant (pro-toxic) chaperone complexes will also be disclosed.

In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors.

The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2, or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possibly also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis of homology to nonplant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released; that is, they are trapped. When expressed in the wild type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared with affinity-purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates or new adaptors. Several of these trapped proteins overaccumulated in clp mutants or were found as interactors for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, the affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II).

PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, ß-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg2+-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg2+ or Mn2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Biophysical and structural characterization of the putative nickel chaperone CooT from Carboxydothermus hydrogenoformans.

Carboxydothermus hydrogenoformans is a model microorganism for the study of [NiFe]-CODH, a key enzyme of carbon cycle in anaerobic microorganisms. The enzyme possesses a unique active site (C-cluster), constituted of a distorted [NiFe3S4] cubane linked to a mononuclear Fe(II) center. Both the biogenesis of the C-cluster and the activation of CODH by nickel insertion remain unclear. Among the three accessory proteins thought to play a role in this latter step (CooC, CooJ, and CooT), CooT is identified as a nickel chaperone involved in CODH maturation in Rhodospirillum rubrum. Here, we structurally and biophysically characterized a putative CooT protein present in C. hydrogenoformans (pChCooT). Despite the low sequence homologies between CooT from R. rubrum (RrCooT) and pChCooT (19% sequence identity), the two proteins share several similarities, such as their overall structure and a solvent-exposed Ni(II)-binding site at the dimer interface. Moreover, the X-ray structure of pChCooT reveals the proximity between the histidine 55, a potential nickel-coordinating residue, and the cysteine 2, a highly conserved key residue in Ni(II)-binding.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells.

The autophagy-lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70-dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA-mediated knockdown of CMA- and mA-related proteins. Knockdown of LAMP2A, a CMA-related protein, and TSG101, an mA-related protein, significantly but only partially decreased the punctate accumulation of GAPDH-HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH-HT puncta in TSG101-knockdown cells, but not in LAMP2A-knockdown cells, suggesting that punctate accumulation of GAPDH-HT under LAMP2A- and TSG101-knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization.

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Immunoreactivity of the AAA+ chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals.

BACKGROUND: Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpBLi) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpBLi as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. RESULTS: ClpBLi consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpBLi and E. coli ClpB is 52 %. The coding sequence of the clpB Li gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpBLi protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpBLi activates the host immune system, as evidenced by an increased level of antibodies against ClpBLi in the sera from infected animals, as compared to the control group. Additionally, ClpBLi was found in kidney tissues of Leptospira-infected hamsters. CONCLUSIONS: ClpBLi is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpBLi point to its potential value as a diagnostic antigen for the detection of leptospirosis.

Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system.

The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.

Crystal structures of Mycobacterial MeaB and MMAA-like GTPases.

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.

Tobacco class I cytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high-temperature stress condition in vitro.

Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon high-temperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, non-denaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200 ∼ 240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures.

[PROTEOMIC ANALYSIS OF ADAPTIVE MECHANISMS TO SALINITY STRESS IN MARINE GASTROPODS LITTORINA SAXATILIS].

Salinity is one of the most important abiotic environmental factors affecting marine animals. If salinity deviate from optimum, adaptive mechanisms switch on to maintain organism's physiological activity. In this study, the reaction of the snails Littorina saxatilis from natural habitats and in response to experimental salinity decreasing was analyzed on proteomic level. The isolation of all snails inside their shells and gradually declining mortality was observed under acute experimental salinity decrease (down to 10 per hundred). Proteomic changes were evaluated in the surviving experimental mollusks compared to control individual using differential 2D gel-electrophoresis (DIGE) and subsequent LC-MS/MS-identification of proteins. Approximately 10% of analyzed proteins underwent up- or down regulation during the experiment. Proteins of folding, antioxidant response, intercellular matrix, cell adhesion, cell signaling and metabolic enzymes were identified among them. Proteome changes observed in experimental hypoosmotic stress partially reproduced in the proteomes of mollusks that live in conditions of natural freshening (estuaries). Possible mechanisms involved in the adaptation process of L. saxatilis individuals to hypo-osmotic stress are discussed.

The crystal structure of the protein-disulfide isomerase family member ERp27 provides insights into its substrate binding capabilities.

About one-third of all cellular proteins pass through the secretory pathway and hence undergo oxidative folding in the endoplasmic reticulum (ER). Protein-disulfide isomerase (PDI) and related members of the PDI family assist in the folding of substrates by catalyzing the oxidation of two cysteines and isomerization of disulfide bonds as well as by acting as chaperones. In this study, we present the crystal structure of ERp27, a redox-inactive member of the PDI family. The structure reveals its substrate-binding cleft, which is homologous to PDI, but is able to adapt in size and hydrophobicity. Isothermal titration calorimetry experiments demonstrate that ERp27 is able to distinguish between folded and unfolded substrates, only interacting with the latter. ERp27 is up-regulated during ER stress, thus presumably allowing it to bind accumulating misfolded substrates and present them to ERp57 for catalysis.

Proteogenomic biomarkers for identification of Francisella species and subspecies by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Francisella tularensis is the causative agent of tularemia. Because some Francisella strains are very virulent, this species is considered by the Centers for Disease Control and Prevention to be a potential category A bioweapon. A mass spectrometry method to quickly and robustly distinguish between virulent and nonvirulent Francisella strains is desirable. A combination of shotgun proteomics and whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry on the Francisella tularensis subsp. holarctica LVS defined three protein biomarkers that allow such discrimination: the histone-like protein HU form B, the 10 kDa chaperonin Cpn10, and the 50S ribosomal protein L24. We established that their combined detection by whole-cell MALDI-TOF spectrum could enable (i) the identification of Francisella species, and (ii) the prediction of their virulence level, i.e., gain of a taxonomical level with the identification of Francisella tularensis subspecies. The detection of these biomarkers by MALDI-TOF mass spectrometry is straightforward because of their abundance and the absence of other abundant protein species closely related in terms of m/z. The predicted molecular weights for the three biomarkers and their presence as intense peaks were confirmed with MALDI-TOF/MS spectra acquired on Francisella philomiragia ATCC 25015 and on Francisella tularensis subsp. tularensis CCUG 2112, the most virulent Francisella subspecies.

Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

[Identification of proteins interacted with Bat3 using tandem affinity purification].

OBJECTIVE: To identify the specific protein interactions involved in Bat3-mediated apoptosis. METHODS: Tandem affinity purification (TAP) was utilized to investigate Bat3-protein interactions, during which full-length human Bat3 fused with Strep2 and FLAG tag as a bait was used to screen the specific protein-protein interactions. The isolated proteins were identified with mass spectrometry. RESULTS: TAP studies showed that Ubl4A was identified as a Bat3-binding partner. Further investigation using co-immunoprecipitation confirmed that Bat3 was associated with Ubl4A. CONCLUSION: TAP was successfully established and is suitable for isolating the binding partners of Bat3.