GST Tag Research Articles

Pichia pastoris as a host for production and isolation of mutagenic AID/APOBEC enzymes involved in cancer and immunity

AID/APOBEC3 enzymes are cytidine deaminases that mutate antibody and retroviral genes and also mediate extensive tumor genome mutagenesis. The study of purified AID/APOBEC3 proteins is challenged by difficulties with their expression and purification arising from genotoxicity in expression hosts, extensive non-specific protein-protein/DNA/RNA interactions and haphazard oligomerization. To date, expression hosts for purification of AID/APOBEC3 enzymes include bacteria, insect and mammalian cells. Here the establishment and optimization of a yeast expression/secretion system for AID/APOBEC3s are reported, followed by comparison with the same enzymes expressed in bacterial and mammalian hosts. AID and APOBEC3G were expressed successfully in Pichia pastoris, each either with an N-terminal GST tag, C-terminal V5-His tag or as untagged native form. It was verified that the yeast-expressed enzymes exhibit identical biochemical properties to those reported using bacterial and mammalian expression, indicating high fidelity of protein folding. It was demonstrated that the system can be adapted for secretion of the enzymes into the media which was used directly in various enzyme assays. The system is also amenable to elimination of bulky fusion tags, providing native untagged enzymes. Thus, P. pastoris is an advantageous expression factory for AID/APOBEC3 enzymes, considering the cost, time, efficiency and quality of the obtained enzymes. The first report is also provided here of a functionally active, untagged, secreted AID, which may become a useful research reagent. A comprehensive comparison is made of the effect of fusion tags and expression hosts on the biochemical actions of AID and APOBEC3G.

EpsM from Bacillus subtilis 168 has UDP-2,4,6-trideoxy-2-acetamido-4-amino glucose acetyltransferase activity in vitro

Bacillus subtilis 168 EpsM (UniProt id P71063) has been electronically annotated as putative acetyltransferase in the UniProt database. The gene epsM was cloned and overexpressed in E. coli with an N-terminal GST tag. The purified fusion protein was shown by absorption spectroscopy, autoradiography and reverse phase HPLC to catalyse the conversion of UDP-2,4,6-trideoxy-2-acetamido-4-amino glucose to UDP-2,4,6-trideoxy-2,4-diacetamido glucose, commonly known as N,N′-diacetylbacillosamine, using acetyl coenzyme A as the donor substrate. His146 was shown by site-directed mutagenesis to be essential for acetyltransferase activity. It is hypothesized that EpsC (NAD+ dependent UDP GlcNAc 4,6-dehydratase), EpsN (PLP dependent aminotransferase) and EpsM, all of which are part of the eps operon, are involved in the biosynthesis of N,N′-diacetylbacillosamine.

Screening and identification of B-cell epitopes within envelope protein of tembusu virus

BackgroundTembusu virus is a newly emerging flavivirus that caused egg-drop syndrome in ducks in China. TMUV envelope protein is a major structural protein locates at the surface of tembusu virus particle. During tembusu virus infection, envelope protein plays a pivotal role in induction of neutralizing antibody. However, B cell epitopes within envelope protein have not been well studied.MethodA series of 13 peptides derived from E protein of tembusu virus were synthesized and screened by Dot blot with tembusu virus-positive duck serum. Potential B-cell epitopes were respectively fused with GST tag and expressed in E. coli. The immunogenicity and protective efficiency of epitopes were assessed in ducks.ResultsDot blot assay identified the peptides P21 (amino acids 301–329), P23 (amino acids 369–387), P27 (amino acids 464–471) and P28 (amino acids 482–496) as potential B-cell epitopes within the envelope protein of tembusu virus. Immunization of prokaryotically expressed epitopes elicited specific antibodies in ducks and the specific antibody elicited by P21, P27 and P28 could neutralized tembusu virus. In addition, protective test suggested that P21 and P27 could completely protect immunized ducks from TMUV challenge.ConclusionFour potential B cell epiotpes within tembusu virus envelope protein were identified and analyzed in vitro and in vivo. It was demonstrated that two of them (P21 and P27) could elicit neutralizing antibodies in ducks and offer complete protection against tembusu virus challenge. This findings will contribute to the development of epitope vaccine for tembusu virus prevention.

Effects of five proteins secreted by Chlamydia trachomatis on phagocytosis of mouse macrophages and dendritic cells

Objective To analyze the effects of five proteins secreted by Chlamydia trachomatis on the phagocytosis of macrophages and dendritic cells derived from bone marrow cells of C3H/HeJ mice. Methods Glutathione S-transferase (GST)-CT311, GST-GIgA, GST-cHtrA, GST-OmcBc and GST-Pgp3 proteins were prepared through an Escherichia coli prokaryotic expression system and purified by GST MagBeads. Chlamydia membrane protein GST-IncA was also prepared as a control. Proteins of interest were obtained by cleaving off GST-tag with PreScission protease.Macrophages (MΦ) and dendritic cells (DC) were prepared from bone marrow cells of C3H/HeJ mice and pretreated with either 100 μg/ml or 500 μg/ml of the above proteins.LPS was used as a control to testify the specificity of the proteins′ functions.Four hours after pretreatment, fluorescent beads were added to culture media to evaluate the changes in phagocytosis with direct immunofluorescence assay. Results LPS and low concentration (100 μg/ml) of these proteins had no significant influence on the phagocytosis of DC and MΦ, while high concentration (500 μg/ml) of Pgp3, cHtrA and CT311 could significantly promote the phagocytosis of DC and MΦ. Conclusion Pgp3, cHtrA and CT311 can promote the in vitro phagocytosis of DC and MΦ, which may facilitate the in vivo dissemination of Chlamyida trachomatis. Key words: Chlamydia trachomatis; Secretory protein; Macrophage; Dendritic cell; Phagocytosis

Covalent and selective immobilization of GST fusion proteins with fluorophosphonate-based probes.

Using GST fusion protein tags is an attractive approach for protein immobilization. Here we report that pyrimidine-based small-molecule probes with a fluorophosphonate reactive group could specifically react with the tyrosine-111 residue of the Schistosoma japonicum GST (sjGST) tag, and these probes could rapidly and site-selectively immobilize sjGST fusion proteins while preserving their activities.

Aptamer facilitated purification of functional proteins

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method’s ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.

Expression, purification, stability and transduction efficiency of full-length SOD2 recombinant proteins

Superoxide dismutase (SOD) family is necessary to protect cells from the toxicity of reactive oxygen species produced during normal metabolism. Among SODs, manganese-containing superoxide dismutase (Mn-SOD, SOD2) is the most important one. The DNA fragment containing the full nucleotide of full-length human SOD2 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with tag GST. DNA construct was then transformed into Escherichia coli BL21 (DE3) and expression was induced with IPTG at 25 ℃. The recombinant fusion protein GST-SOD2 (46 kDa) was purified from the bacterial lysate by GST resin column affinity chromatography. GST tag was cleaved with thrombin, and a crude SOD2 recombinant protein (25 kDa) was obtained and further purified by heparin affinity chromatography. Activities of the two SOD2 proteins were 1 788 and 2 000 U/mg, respectively. Both SOD2 proteins were stable under physiological condition and cell-penetrating (P<0.05). Our findings open the possibility to study the structure and effects of two full-length recombinant SOD2 proteins.

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected “Universal-IPCR” format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.

Chaperone-assisted maturation of the recombinant Fe-type nitrile hydratase is insufficient for fully active expression in Escherichia coli

Nitrile hydratase (NHase) has attracted substantial attention for industrial applications to produce large-scale amides. Several NHases have been investigated for functional expression in Escherichia coli (E. coli). A Fe-type NHase was obtained from an acetamiprid-degrading bacterium, Pseudoxanthomonas sp. AAP-7 and functionally expressed in E. coli BL21 (DE3). No significant NHase activity was detected from the E. coli expressing either the NHase gene alone or NHase and P46K genes transcribed as one unit. Purified recombinant NHase, co-expressed with P46K on two separate plasmids, exhibited the maximal enzyme activity. Furthermore, a GST tag attached to the N-terminus of α subunit resulted in a slight increase in the solubility and stability of NHase compared with a His tag at the C-terminus of β subunit. When co-expressed with the chaperones GroEL-GroES, the yield of the soluble recombinant NHase was improved substantially, while a small decrease in NHase activity was observed. The putative activator P46K was strictly required for production of the recombinant NHase for full enzyme activity, although the chaperones GroEL-GroES appeared to assist NHase to fold properly. This study of the expression of a fully active Fe-type NHase would provide another example to enhance our understanding of NHase biosynthesis.

Auxin Extraction and Purification Based on Recombinant Aux/IAA Proteins

BackgroundIndole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones.ResultsSince the AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) proteins show higher specific binding capability with auxin, recombinant IAA1, IAA7 and IAA28 proteins were used as sorbents to develop an IAA extraction and purification method. A GST tag was used to solidify the recombinant protein in a column. Aux/IAA proteins solidified in a column have successfully trapped trace IAA in aqueous solutions. The IAA7 protein showed higher IAA binding capability than the other proteins tested. In addition, expression of the IAA7 protein in Drosophila Schneider 2 (S2) cells produced better levels of binding than IAA7 expressed in E. coli.ConclusionThis work validated the potential of Aux/IAA proteins to extract and purify IAA from crude plant extracts once we refined the techniques for these processes.

Subtilisin-like serine protease gene TghSS42 from Trichoderma ghanense ACCC 30153 was successfully expressed in Escherichia coli and recombinant protease rTghSS42 exhibited antifungal ability to five phytopathogens.

The subtilisin-like serine protease gene TghSS42 was cloned from biocontrol agent Trichoderma ghanense ACCC 30153. Its coding region is 1302 bp in length, encoding 433 aa with a predicted protein molecular weight of 42.5 kDa and pI of 5.53. The accession number of cDNA sequence of TghSS42 gene is KJ740359. Furthermore, the transcription of the TghSS42 gene was all up-regulated under nine different treatments by RT-qPCR analysis, and the highest transcription level of TghSS42 approached 177.29-fold at 4 h under induction with 1% (w/v) Alternaria alternata cell walls, indicating that TghSS42 could be induced by the plant or phytopathogen. Furthermore, Escherichia coli recombinant strain BL21-TghSS42 was constructed. The recombinant protease rTghSS42, with an expected molecular weight of approximately 68.5 kDa (containing 26.0 kDa GST tag), has been successfully expressed and purified from BL21-TghSS42. The purified protease rTghSS42 activity reached a peak of 18.7 U/mL at 4 h following 1.0 mM IPTG induction. The optimal enzyme reaction temperature was 40℃ and the optimal pH was 7.0. The recombinant protease rTghSS42 exerted broad-spectrum antifungal ability against Rhizoctonia solani, Fusarium oxysporum, A. alternata, Sclerotinia sclerotiorum and Cytospora chrysosperma. The inhibition rate of mycelial growth varied between 21.2% and 50.0%.

Production of Polyclonal Antibody to the HPV58 E7 Protein and Its Detection in Cervical Cancer.

The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer worldwide, and HPV type 58 is the third most common HPV type in eastern Asia. The E7 oncoprotein is constitutively expressed in HPV58-associated cervical cancer cells and plays a key role during tumorigenesis. To study the biological function of HPV58 E7 and to characterize E7 protein-host cell interactions, we cloned the human HPV58 E7 gene and produced specific E7 antibodies. The HPV58 E7 gene was cloned into a prokaryotic expression vector, pGEX-4T2. The recombinant plasmid pGEX-4T2-(HPV58-E7) was transformed into Escherichia coli DH5α and expressed as a fusion protein containing a GST tag. After purification and removal of the GST affinity tag, the E7 protein was used as an antigen for the production of antiserum in rabbits. The specificity of the purified HPV58 E7 antibody was detected by western blotting, immunofluorescence and immunohistochemistry analysis. These methods demonstrated that the polyclonal antibody could specifically recognize the endogenous and the recombinant HPV58 E7 proteins. Immunohistochemistry analysis indicated that the E7 protein was localized in the nucleus of cervical cancer cells.

Multifaceted analyses of the interactions between human heart type fatty acid binding protein and its specific aptamers

BackgroundAptamer-protein interaction studies have been mainly confined to dissociation constant (Kd) determination. A combinatorial approach involving limited proteolysis mass spectroscopy, molecular docking and CD studies is reported here to elucidate the specific interactions involved. MethodsTo generate aptamers specific for human FABP3, SELEX was performed incorporating counter SELEX cycles against control FABPs and GST tag, followed by their characterization by EMSA, CD and SVD analysis. Based on computationally obtained aptamer-protein complex models, the interacting aptamer, and protein residues were predicted and supported by limited proteolysis experiments. ResultsTwo aptamers N13 and N53 specific for human fatty acid binding protein (FABP3) were isolated with corresponding Kd of 0.0743±0.0142μM and 0.3337±0.1485μM for FABP3 interactions. Both aptamers possess stable B-DNA structures at salt concentration of 100mM and pH range (6–9). The N13 aptamer led interaction involved 3 salt bridges and 2 hydrogen bonds, whereas N53 had 2 salt bridges with 8 hydrogen and 7 hydrophobic interactions. ConclusionsThe aptamers generated are the first to be reported against human FABP3. The higher interaction footprint of N53 incited synergistic conformational changes in both N53 and FABP3 during interaction, leading to a decline in binding affinity in comparison to N13 which corroborated to the calculated Kd values. General significanceThis combinatorial method may be used to retrieve the possible specific binding modes and interaction patterns involved in large aptamer-protein complexes. Thus the method can be exploited to identify the optimum aptamer length for in-depth structure-function studies and its tailored applications.

Engineering of a novel carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis of enantiopure chiral alcohols

The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.

Successful expression of the Bordetella petrii nitrile hydratase activator P14K and the unnecessary role of Ser115.

BackgroundThe activator P14K is necessary for the activation of nitrile hydratase (NHase). However, it is hard to be expressed heterogeneously. Although an N-terminal strep tagged P14K could be successfully expressed from Pseudomonas putida, various strategies for the over-expression of P14K are needed to facilitate further application of NHase.ResultsP14K was successfully expressed through fusing a his tag (his-P14K), and was over-expressed through fusing a gst tag (gst-P14K) at its N-terminus in the NHase of Bordetella petrii DSM 12804. The stability of gst-P14K was demonstrated to be higher than that of the his-P14K. In addition, the Ser115 in the characteristic motif CXLC-Ser115-C of the active center of NHase was found to be unnecessary for NHase maturation.ConclusionsOur results are not only useful for the NHase activator expression and the understanding of the role of Ser115 during NHase activation, but also helpful for other proteins with difficulty in heterologous expression.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori.

β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

Abstract 1318: A protein microarray signature of autoantibody biomarkers for the detection of HER2+ breast cancers

Abstract Introduction: Breast cancer is a heterogeneous disease, clinically defined by expression of the biomarkers ER, PR, and HER2, with 15-30% of breast cancers overexpressing the HER2 protein. Antibodies to tumor antigens that are present in patient sera are potential biomarkers for early detection and prognosis of breast cancer. The purpose of this study is to identify a panel of novel serum biomarkers for the early diagnosis of HER2+ breast cancer. Methods: We screened sera from breast cancer patients for autoantibodies using custom nucleic acid programmable protein microarrays (NAPPA). Plasmid cDNAs encoding 10,000 full-length human proteins with c-terminal GST or FLAG tags were printed on arrays with anti-GST or anti-FLAG antibodies, and transcribed and translated in situ using HeLa expression lysate. Serum samples were obtained at diagnosis from patients with stage I-III HER2+ breast cancer classified by IHC and/or FISH testing (HER2+/ER-/PR-, 47%; HER2+/ER+/PR+, 48%; HER2+/ER-/PR+, 5%). Control sera were age-matched and obtained from women undergoing screening mammography or diagnostic mammography for benign breast disease. The microarrays were screened with sera from 29 cases and 29 controls. 850 antigens were selected, displayed in microarray format, and screened with an independent set of sera 27 cases, 29 benign breast disease controls, and 25 healthy controls. Bound IgG was detected by fluorescence. Results: From the initial 10,000 proteins, we identified 850 proteins using cutoff values of AUC (0.68), sensitivity (0.29), and pAUC (0.0075) at 94% specificity for further analysis. From these, 50 candidate autoantibody biomarkers for the detection of HER2+ breast cancer were identified based on visual analysis and statistical cutoff values of 15% sensitivity at 98% specificity and K-statistic (≥ 0.9). Conclusion: We identified a 50-autoantibody serum signature for the detection of HER2+ breast cancer. Blinded validation studies are ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Matthew S. Field, Jennifer Viloria, Garrick Wallstrom, Joshua LaBaer, Paul Engstrom, Karen S. Anderson. A protein microarray signature of autoantibody biomarkers for the detection of HER2+ breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2015-1318