Protease Cleavage Sequence Research Articles

Synthetic protein protease sensor platform.

Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.

Principles to recover copper-conducting CTR proteins for the purpose of structural and functional studies

Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.

Enhanced Zika virus-like particle development using Baculovirus spp. constructs.

Zika virus (ZIKV) is one of several examples of an unprecedented pandemic spread and against which there is currently no suitable vaccine or treatment.Here, we constructed and characterized recombinant baculovirus-derived ZIKV-like particles (Zika VLPs) to study ZIKV-antibody interactions. These VLPs, uniquely consisted of the full-length ZIKV capsid (C), pre-membrane (prM), and envelope (E) proteins with either: a) the viral nonstructural NS2B and NS3 protease unit under one or two different promoters or b) an alternative host-cell furin protease encoding cleavage sequence inserted between the C and prM genes, together with lobster tropomyosin leader and honeybee signal sequences with one promoter for increased extracellular secretion.All these Zika VLPs displayed typical virion morphology in transmission electron microscopicanalysis when expressed in both insect (Sf9) and mammalian (HEK293T) cells and no uncleaved prM glycoprotein was detected, as are present on immature virions. The importance of glycosylation of the E glycoprotein was shown by the effects on both polyclonal and monoclonal antibody reactions after these N-linked carbohydrate residues were disrupted by oxidation or enzymatic cleavage.Importantly, the construct which contained the host-cell furin protease cleavage sequence together with a lobster tropomyosin leader and honeybee signal sequences under one promoter produced higher Zika VLP titers and protein concentrations and which can now be tested as a superior construct in multifunctional diagnostic (ELISA and neutralization/antibody-dependent enhancement) assays and immunogenic assessments possibly leading to vaccine trials.

Antibody-Peptide Epitope Conjugates for Personalized Cancer Therapy.

This study develops a high-throughput preclinical platform to identify patient-specific antibody-peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner.

The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1–0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, includingneo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Development of an Interleukin-12 Fusion Protein That Is Activated by Cleavage with Matrix Metalloproteinase 9.

Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cell-mediated responses and can enhance antitumor responses in experimental models. IL-12 has been tested clinically, however, side-effects have limited its use. We are developing an attenuated form of IL-12 whose biological activity could be restricted to sites of tumors by taking advantage of overexpressed tumor proteases that can activate the cytokine. We constructed a panel of fusion proteins (FPs) consisting of IL-12 joined to a specific inhibitor connected by a protease cleavage sequence (cs). We first identified a panel of single-chain Fragment variable (scFv) that bind to 3 independent epitopes on IL-12 and then incorporated them into separate IL-12 FPs containing either a matrix metalloproteinase (MMP) cs or a scrambled (scram) control cs. The intact IL-12 FPs showed attenuation in IL-12 activity compared to free IL-12 in 2 separate in vitro functional assays; proliferation of CTLL-2 and interferon-gamma (IFN-γ) induction by spleen cells. Furthermore, the FP containing the MMPcs showed an increase in biological activity of IL-12 in vitro when cleaved by MMP9. This FP strategy could be applied to other immunomodulators and potentially reduce unwanted side-effects observed with systemic delivery thus improving cytokine immunotherapy strategies.

Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors.

Protease-activatable viruses (PAV) based on adeno-associated virus have previously been generated for gene delivery to pathological sites characterized by elevated extracellular proteases. "Peptide locks", composed of a tetra-aspartic acid motif flanked by protease cleavage sequences, were inserted into the virus capsid to inhibit virus-host cell receptor binding and transduction. In the presence of proteases, the peptide locks are cleaved off the capsid, restoring the virus' ability to bind cells and deliver cargo. Although promising, questions remained regarding how the peptide locks prevented cell binding. In particular, it was unclear if the tetra-amino acid (4AA) motif blocks receptor binding via electrostatic repulsion or steric obstruction. To explore this question, we generated a panel of PAVs with lock designs incorporating altered 4AA motifs, each wielding various chemical properties (negative, positive, uncharged polar, and hydrophobic) and characterized the resultant PAV candidates. Notably, all mutants display reduced receptor binding and decreased transduction effciency in the absence of proteases, suggesting simple electrostatics between heparin and the D4 motif do not play an exclusive role in obstructing virus-receptor binding. Even small hydrophobic (A4) and uncharged polar (SGGS) motifs confer a reduction in heparin binding compared to the wild type. Furthermore, both uncharged polar N4 and Q4 mutants (comparable in size to the D4 and E4 motifs respectively, but lacking the negative charge) demonstrate partial ablation of heparin binding. Collectively, these results support a possible dual mechanism of PAV lock operation, where steric hindrance and electrostatics make nonredundant contributions to the disruption of virus-receptor interactions. Finally, because of high virus titer production and superior capsid stability, only the negatively charged 4AA motifs remain viable design choices for PAV construction. Future studies probing the structure-function relationship of PAVs will further expand its promise as a gene delivery vector able to target diseased tissues exhibiting elevated extracellular proteases.

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses.

Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades.

Development of protease activated interleukin-12 cytokine fusion proteins for tumor immunotherapy (TUM7P.946)

Abstract Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.

A new fusion protein platform for quantitatively measuring activity of multiple proteases

BackgroundRecombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases.ResultsWe tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract.ConclusionThe results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity.

GFP Variants with Alternative Strands: Protease Sensor Design and their Thermodynamic Analysis

Green fluorescent protein (GFP) variants with one extra strand 10 (s10) can fold with either one or the other s10, and the composition of the two bound forms can be unambiguously distinguished by their resulting colors (Do & Boxer, 2013 JACS). The composition can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have applied thermodynamic analysis on the composition to obtain insight into folding of the variants, and discovered robust empirical rules that accurately predict the product ratios of a given construct. Ratiometric protease sensors were designed from the construct by recombinantly adding a protease cleavage sequence into one of the inserted loops, where the bound s10 is replaced by the other upon protease cleavage and irradiation with light, which is based on a photodissociation phenomenon previously reported (Do & Boxer, 2011 JACS). Since the conversion between the two bound forms involves a large spectral shift, the sensor can display a very high dynamic range. The variants can serve as a platform to provide useful insight into protein folding in general, and further engineering of this class of proteins will enable their application as light-dependent modulators.

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells

BackgroundRicin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA. MethodsHIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants. ResultsC-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells. ConclusionsRTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication. General significanceCurrent cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV.

Ahp Cyclodepsipeptides: The Impact of the Ahp Residue on the “Canonical Inhibition” of S1 Serine Proteases

S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small-molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small-molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small-molecule inhibitors that simultaneously target the S and S' subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β-hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.

Expression pattern of recombinant organophosphorus hydrolase from Flavobacterium sp. ATCC 27551 in Escherichia coli

Concerned with the influence of tagging system on the expression of heterogeneous protein in Escherichia coli, we attempted to express the organophosphorus hydrolase (OPH) of Flavobacterium sp. ATCC 27551 in E. coli. Recombinant OPH was overproduced successfully in E. coli when modified without the use of a tobacco etch virus (TEV) protease cleavage sequence. In addition, though there has never been a report on the extracellular secretion of recombinant OPH harboring native Tat signal peptides in E. coli, the produced protein was observed to be secreted extracellularly. Through the use of reverse transcriptional quantitative real-time PCR and comparison of the predicted folding rate, it was determined that OPH expression may be affected by the existence of a TEV protease cleavage sequence at the C-terminus during the process of translated protein folding, leading to the suppressed OPH activity. With the potential compatibility between native Tat signal peptides of OPH and E. coli Tat pathway secretion system, we report a successful expression of recombinant OPH harboring native Tat signal peptides in E. coli, for the first time.

Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14

An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.

Protease activated interleukin-2 cytokine fusion proteins for tumor immunotherapy (46.17)

Abstract Cytokines are being used for tumor immunotherapy, but when delivered systemically, negative side effects limit their utility. To mitigate these side effects but maintain their anti-tumor effects, we have constructed a panel of fusion proteins (FPs) with IL-2 joined to a specific inhibitor, separated by specific protease cleavage sequences. The rationale is that at the tumor site, where the concentration of a protease is high, the FP will be cleaved, allowing the IL-2 to dissociate from the inhibitor, increasing the biological activity of the cytokine and enhancing anti-tumor effectors. We have expressed these FPs and showed that cleavage in vitro results in increased biological activity of IL-2. In the Colon 38 peritoneal tumor model, delivery of a FP results in increased frequency of T cells (p< 0.0001) and NK cells (p< 0.01) compared to vehicle control treatment, and a corresponding decrease in tumor growth. To test for systemic side effects and evaluate the effects of FP delivery, we used an adeno-associated virus (AAV) delivery system to achieve sustained, high systemic levels of the FPs in mice. AAV-expressed FPs persist at high levels (~500 pg/ml) for at least 9 weeks and do not appear immunogenic. Importantly, unlike injection of free IL-2, expression of the FPs did not generate an inflammatory cytokine response as determined by a 22-analyte Luminex assay. Thus, protease activated cytokine fusion proteins may be a valuable means to enhance tumor immunotherapy.

Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.