C-myc Tag Research Articles

In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv’s structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

Impaired malin expression and interaction with partner proteins in Lafora disease

Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin invivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making an expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and several glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/laforin knockout and malin-myc/LaforinCS, where laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires laforin but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that laforin and malin form a complex invivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.

Serum Western Blot for the Detection of a c-Myc Protein Tag in Non-human Primates and Mice.

This protocol allows for the detection of a c-Myc tag on alpha-1 antitrypsin (AAT) delivered to species that already have endogenous AAT such as non-human primates allowing reliable and repeatable semi-quantitation of serum levels of AAT.

A high affinity and specificity anti-HER2 single-domain antibody (VHH) that targets trastuzumab's epitope with versatile biochemical, biological, and medical applications.

In the past decade, various single-domain antibodies from llamas, also known as VHH or nanobody, have been discovered with applications in tumor imaging and cancer therapy. However, the potential application of anti-HER2 VHHs as a diagnostic tool suitable for ELISA, flow cytometry, cell imaging, bispecific antibody engineering,and immunohistochemistry has not been fully elucidated. To investigate this potential, HER2 antigen was expressed in HEK293 F cells, purified, and used to immunize llama. Using phage display, anti-HER2 VHHs with high affinity and specificity were isolated, sequenced, and constructed with a Histag and c-Myc tag. The constructed anti-HER2 VHHs were then expressed in E. coli, purified, and evaluated for their use in ELISA, flow cytometry, cell imaging, and immunohistochemistry. The affinities of the anti-HER2 VHHs toward the HER2 antigen were determined using biolayer interferometry. Furthermore, the binding sites of the anti-HER2 VHHs were evaluated by epitope mapping and in silico modeling and docking. Here, we report the sequence of an anti-HER2 VHH with high affinity (sub-nanomolar), specificity, and selectivity. This VHH binds to the same epitope as trastuzumab and can be utilized to generate bispecific antibodies or used as a diagnostic tool to differentiate HER2+ from HER2- antigens on plates, cells, and tissues. This discovery has broad applications in biochemical, biological, and medical sciences.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector.

The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.

Non-negligible factors in studying the ApoM-S1P axis using EA.hy926 cells.

BackgroundThe apolipoprotein M (ApoM)-sphingosine-1-phosphate (S1P) axis was recently identified, and research into its function has received increasing attention. However, there are some factors which might influence the results of studies into the function of the ApoM-S1P axis using the EA.hy926 cells. This study investigated related factors, including coagulation factor VIII (FVIII), ApoM, S1P receptor subtypes (S1PRs), C-myc-tagged, and His-tagged proteins in EA.hy926 cells, as well as the effects of ApoM overexpression on S1PRs.MethodsThe expression of FVIII, ApoM, S1PRs, C-myc, and His-tagged proteins in EA.hy926 cells was investigated through cellular immunofluorescence. EA.hy926 cells were infected with lentiviruses carrying (OE group) or lacking (NC group) the ApoM gene sequence. A stable cell line expressing ApoM was obtained, and the expression of ApoM mRNA was detected through single tube duplex fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR). S1PRs expression was detected by RT-qPCR and Western blotting.ResultsThe results showed that EA.hy926 cells expressed FVIII, ApoM, C-myc-tagged, and His-tagged proteins. Moreover, they highly expressed S1PR1, slightly expressed S1PR3, weakly expressed S1PR2, and did not express S1PR4 and S1PR5. ApoM overexpression significantly increased S1PR1 mRNA and protein expression but did not affect the expression of S1PR3. EA.hy926 cells expressed FVIII, suggesting the cell line possesses endothelial cell characteristics and could be used for in vitro studies of the ApoM-S1P axis.ConclusionsEA.hy926 cell line is suitable for investigation of the ApoM-S1P axis in vitro. However, Since EA.hy926 cells expressed endogenous ApoM, C-myc and His tagged proteins, the exogenous recombinant ApoM should not be labeled with C-myc and His tags for distinguishing from endogenous ApoM. In addition, overexpression of ApoM should be considered to significantly increase the expression of S1PR1 when studying the APOM-S1P axis.

Enterotoxins can support CAR T cells against solid tumors

Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vβ subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2–3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 μg/mL with VLP versus 38 μg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

The Inhibition of Gck Affect MAPK Cascade in Multiple Myeloma

Introduction: Germinal Center Kinase (GCK) is an essential regulator of stress-activated MAPK core signaling pathways. Recent studies revealing the role of GCK in tumorigenesis and cancer progression have generated increasing interest in validating GCK as a therapeutic target in cancer. We previously showed that GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation (Li et al, Blood 2017 130:1795). Our further research established GCK as a critical kinase in MM and as an attractive target for treatment of MM.Methods and Results: The over-expression of GCK in primary MM compared to normal cells was confirmed by immunohistochemistry staining of primary MM patient bone marrow biopsies. By quantitatively comparing the staining scores of GCK of primary MM patient bone marrows (n=26) and normal donor bone marrows (n=26), we found a significantly higher GCK expression (p<0.001) in MM patient BM samples (98%) compared to healthy donor BM samples (47%) (Figure 1). GCK protein expression level was induced by several cytokines that are critically involved in MM cell survival and proliferation including IL-6, TNF-a, LPS and CD40L in MM cells after treatment for 1h. The role of GCK expression in MM signaling transduction and tumor progression was addressed by inducible GCK-knockdown in MM cells. By generating Tet-on-shGCK-MM cell lines, we found that doxycycline-induced GCK silencing blocks IL-6 induced phosphorylation of MKK4, MKK7, JNK and ERK in MM.1S cells. Moreover, knockdown of GCK causes downregulation of IKZF1/3, c-MYC and BCL-6 in MM cells.To exclude the off-target effects of the GCK-shRNA and confirm the specific and critical role of GCK, we further generated recombinant GCK-WT and GCK-shRNA resistant allele construct with C-terminal c-Myc tag, and introduced them into the GCK inducible KD cells to determine if shRNA-resistant GCK can rescue the growth inhibition induced by the shRNA. Tet-on-shGCK-RPMI-8266 cells transduced with empty vector (EV) or GCK wild-type with c-Myc tag (WT) or GCK shRNA resistant mutation with c-myc tag (MT) were generated by routine lentiviral infection. Doxycycline-induced tet-on-shGCK expression resulted in significant decrease of GCK protein. Both endogenous GCK and wild-type GCK-c-Myc expressions were silenced. In contrast, expression of GCK shRNA resistant allele with c-Myc tag (MT) was not affected. The rescue effects of this allele on MM cells growth inhibition are undergoing.Bay 61-3606 is a small molecule that selectively inhibits GCK activity and shows promising anti-MM effects in our previous in vitro and in vivo studies. To further confirm the mechanism of GCK inhibition, we examined its effects on the MAPK cascade and found that pharmacologic blockade of GCK by Bay 61-3606 dose-dependently downregulated ERK, p38 and JNK phosphorylation in MM cells.In MM, IMiDs treatment leads to the selective ubiquitination and degradation of IKZF1 and IKZF3 by the CRBN-CRL4 ubiquitin ligase. Suppression of CRBN in MM cell lines induces IMiDs resistance. To our surprise, in MM cells with CRBN-knockdown, Bay 61-3606 retained the ability to decrease IKZF1 protein level and inhibit MM cell growth, indicating that GCK-induced IKZF1 downregulation is independent of CRBN. Thus, this study further validates the therapeutic potential of GCK inhibitors in IMiDs-resistant MM.Conclusion: Our findings show that GCK is overexpressed in MM and an attractive target for multiple myeloma therapy. The inhibition of GCK blocks the phosphorylation and activation of MAPK cascade, and induces the degradation of the substrate proteins IKZF1, IKZF3 and c-Myc, (Figure 2.). These studies further confirm the critical role of GCK in MM tumorigenesis. GCK inhibitors represent a novel anti-MM therapy especially in relapsed and IMiDs-resistant MM patients. [Display omitted] DisclosuresLentzsch:Caelum Biosciences: Consultancy, Other: Dr. Lentzsch recused herself as an investigator from the Phase 1a/b trial testing CAEL-101 in 11/2017., Patents & Royalties: Shareholder for Caelum Biosiences; Janssen: Consultancy; Bayer: Consultancy; BMS: Consultancy.

Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5’-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine inBacillus subtilis

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L-1 and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.

Abstract 5764: Discovery of therapeutic human monoclonal antibody combinations using microfluidics and molecular genomics

Abstract Monoclonal antibodies (mAbs) have proven to be extremely effective immuno-oncology therapeutics that lead to previously unseen durable responses in melanoma, bladder cancer, RCC, lung cancer, and other malignancies. Most mAb therapeutics, including current anti-PD-1 drugs, were discovered by mouse immunization followed by hybridoma isolation. Unfortunately, mouse hybridomas are highly inefficient, capturing only about 0.1% of the total B-cell diversity in immunized mice. As a result, discovery programs that use mouse hybridomas often struggle to find high-affinity, developable antibodies against certain challenging targets, even when using humanized mice. We have developed a novel method for mouse mAb discovery that leverages microfluidics, multiplex PCR, yeast single chain variable fragment (scFv) display, and fluorescence-activated cell sorting (FACS). Our technology is significantly faster and more comprehensive than conventional hybridoma screening. We recently embarked on a discovery campaign for 17 immuno-oncology targets, including PD-1, CTLA-4, GITR, and OX40. We injected chimeric humanized mice (Trianni) with antigen twice weekly for three weeks. Lymph nodes, splenocytes, and bone marrow were removed from the mice and made into single-cell suspensions. We then isolated B cells using negative selection magnetic beads, and used microfluidics to process &amp;gt;4 million cells per mouse, generating DNA libraries that keep single cell heavy and light chain immunoglobulin (Ig) pairing intact. We built yeast scFv libraries for each of the 17 antigen-specific B cell repertoires with a C-terminal c-myc tag. To select scFv-expressing cells that bind to antigen, biotinylated antigen was added to the yeast cells at 7-250nM and then stained with fluorescent streptavidin. We then flow sorted for the top double-positive cells (c-myc+/antigen+). Finally, we deep sequenced the libraries of enriched high-affinity scFvs, which yielded the sequences for &amp;gt;2,000 candidate scFv binders. Hundreds of these candidate scFvs were engineered into full-length mAb constructs and expressed transiently in Chinese hamster ovary (CHO) cells. To develop candidates for clinical use, we performed assays for binding kinetics, cell surface antigen binding, in vitro T-cell activation, and epitope binning. Candidates that are high affinity, bind cell surface antigen, and induce immune cell activation are now being tested in combinations for in vitro and in vivo efficacy. Combinations that have significantly better in vitro and in vivo efficacy than benchmark checkpoint inhibitors currently on the market will be moved forward into clinical studies. Citation Format: David S. Johnson, Adam S. Adler, Rena Mizrahi, Carter Keller. Discovery of therapeutic human monoclonal antibody combinations using microfluidics and molecular genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5764.

Ileum-Specific Transgenic Expression of Human Chimeric Intestinal Alkaline Phosphatase (IAP) Attenuates Western Diet (WD)–Induced Barrier Dysfunction and Glucose Intolerance

Intestinal epithelial cell derived IAP dephosphorylates/detoxifies bacterial endotoxin LPS in the gut lumen. We have earlier demonstrated that improvement of intestinal barrier function was accompanied by an increase in IAP activity. Herein, we developed IAP transgenic mice where expression of human chimeric IAP is under the control of intestine specific villin promoter. As shown in Panel A, compared to non-transgenic wild type (WT) littermates, IAP expression (identified by c-myc tag) was noted in colon, ileum jejunum and duodenum of IAP-transgenic (IAPTg) mice. IAP was almost uniformly expressed along the length of the ileum (Panel B) and while IAP activity reduced from proximal P1 segment to distal P9 segment in WT mice, this activity was maintained in the IAPTg mice (Panel C). Dietary challenge with WD impaired glucose tolerance in WT mice and this intolerance was attenuated in IAPTg mice (Panels D and E). Significant decrease in fecal zonulin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice (Panel F) and a corresponding decrease in translocation of oral 4kDa FITC dextran to plasma (Panel G) suggests that IAP overexpression improves intestinal barrier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD induced intestinal dysfunction and glucose intolerance. Disclosure H. He: None. S.S. Ghosh: None. P.J. Yannie: None. J. Wang: None. S. Ghosh: None.

Cardiac cell modelling of myofibrillar myopathy by AAV-mediated transduction of desmin mutations in human iPSC-derived and neonatal rat cardiomyocytes

Myofibrillar myopathy (MFM) is a group of severe cytokeletal proteins known to be caused by mutations in genes encoding. Among the myofibrillar proteins that are accumulated within the muscle fibers of affected patients, they appear to be responsible for the structural integrity of striated muscle. Mutations in the gene lead not only to skeletal muscle myopathies but also to cardiomyopathies, however the cardiac pathological consequences caused by them remain poorly understood. The objective of this work is to examine the effects of MFM-induced mutations in the structural organization of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) as identified by troponinT immunostaining, and rat neonatal cardiomyocytes. We propose an in vitro cardiac disease-based cellular model based on adeno-associated virus (AAV)-mediated transduction of wild-type (WT) and mutated desmin in fusion with a c-myc tag to distinguish between endogenous and transgenic desmin proteins. Our preliminary results demonstrated that cardiomyocytes were successfully transduced by AAV vectors and expressed exogenous WT desmin as revealed by c-myc immunostaining. For better recapitulating the in vivo anisotropic cardiomyocyte organization, we have developed micro-patterned culture plates made by microcontact printing where thin strips of gelatin have been deposited on top of glass. Culture of both hiPSC-derived and rat neonatal cardiomyocytes on this system improved their sarcomere alignment. The analysis based on immunostaining of protein-markers to examine structural organization in AAV-transduced cardiomyocytes is in progress. Our results indicate that the proposed in vitro model is suitable for the study of the structural defects of desmin-mutated cardiomyocytes.

Investigation of multivalent interactions between conjugate of quantum dots with c-Myc peptide tag and the anti-c-Myc antibody by capillary electrophoresis with fluorescence detection.

Herein, we report an assay for detecting the binding of a multivalent peptide and antibody within a capillary with the use of fluorescence coupled capillary electrophoresis. Quantum dots and a c-Myc tag containing peptide EQKLISEEDLG4 H6 were injected sequentially and formed a multivalent quantum dot-EQKLISEEDLG4 H6 assembly within the capillary. The efficiency of the quantum dot-peptide self-assembly was affected by the peptide/quantum dot molar ratio, sampling time, and interval time. Finally, the binding of the monoclonal anti-c-Myc antibody and the multivalent quantum dot-EQKLISEEDLG4 H6 ligand was studied using an in-capillary assay. The microscopic dissociation constant for the self-assembly of monoclonal anti-c-Myc antibody and quantum dot-EQKLISEEDLG4 H6 was determined to be 14.1 μM with a stoichiometry of the peptide-antibody complex of 1.7 determined after fitting this to the Hill equation. This method can be further extended to detect a wide range of biomolecule-biomolecule binding interactions.

Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P.pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P.pastoris transformant. Highest hyaluronidase activity of 2mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100-300kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC50) of 8mM against rHuPH20.

Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins

The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins.

Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles

Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris

Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75mg/L rhIL-15 after 3days of induction by methanol. The rhIL-15 was purified by Ni+-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from P. pastoris.

Prion protein "gamma-cleavage": characterizing a novel endoproteolytic processing event.

The cellular prion protein (PrP(C)) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrP(C) is recognized to undergo both α- and β-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrP(C), as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrP(C) fragment, nominally 'C3', and elaborating existing nomenclature, 'γ-cleavage' as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrP(C) has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrP(C) species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrP(C) processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration.