Cleavage Of Fusion Protein Research Articles

Comparison of cell‐based and cell‐free protocols for producing target proteins from theArabidopsis thalianagenome for structural studies

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.

Characterization of a recombinant granzyme B derivative as a “restriction” protease

Blood coagulation factor X a (FX a) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases. We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FX a and Thrombin as processing protease. This recombinant protease is derived from human granzyme B (GrB). The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FX a in two cases.

Expression, refolding, and purification of recombinant human granzyme B

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor X a (FX a) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni 2+–NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FX a or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.

Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.

Botulinum neurotoxins (BoNTs) act as zinc-dependent endopeptidases that cleave proteins required for neurotransmitter release. To detect toxin activity, fragments of the toxin substrate proteins, synaptobrevin (Syb) or synaptosome-associated protein of 25 kDa (SNAP-25), were used to link cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP). Cleavage of these fusion proteins by BoNTs abolished fluorescence resonance energy transfer between the CFP and YFP, providing a sensitive means to detect toxin activity in real-time in vitro. Furthermore, using full-length SNAP-25 and Syb as the linkers, we report two fluorescent biosensors that can detect toxin activity within living cells. Cleavage of the SNAP-25 fusion protein abolished fluorescence resonance energy transfer between CFP and YFP, and cleavage of Syb resulted in spatial redistribution of YFP fluorescence in cells. This approach provides a means to carry out cell-based screening of toxin inhibitors and to study toxin activity in situ. By using these biosensors, we found that the subcellular localizations of SNAP-25 and Syb are critical for efficient cleavage by BoNT/A and B, respectively.

Subcellular localization and calcium and pH requirements for proteolytic processing of the Hendra virus fusion protein.

Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK/L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl3 or incubation of cells at 20 degrees C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca(2+)-dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca2+ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H+ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca2+ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization.

Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of alpha-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His6 encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EKL-His6 was attained by methanol induction and its molecular weight is 43 kD. Because of the existence of His6-tag, EKL-His6 was easily purified from P. pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EKL-His6 demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.

Direct production of proteins with N-terminal cysteine for site-specific conjugation.

Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.

Expression cloned cDNA for 10-deacetylbaccatin III-10- O-acetyltransferase in Escherichia coli: a comparative study of three fusion systems

10-Deacetylbaccatin III-10- O-acetyltransferase (10-DABT) catalyzes the formation of baccatin III, which is an immediate diterpenoid precursor of Taxol. A cDNA encoding 10-DABT was cloned from Taxus baccata by using RT-PCR and screening a cDNA library. A study of its heterologous overexpression in Escherichia coli was carried out. To get high-level expression of recombinant enzyme, three kinds of IPTG inducible fusion expression systems (with glutathione S-transferase (GST), hexahistidine (6× His), and biotinylated tag) were used, and results of expression were compared. Fusion 10-DABT with different tags was expressed with diverse expression levels and solubility in the three systems. Optimum IPTG concentration, temperature, and inducing time for producing recombinant enzymes were found. Under higher IPTG concentration (up to 1 mM), the highest level of expression for fusion protein was obtained in the 6× His fusion system with phage T5 promoter, but expressed products were only partially soluble. With lower IPTG concentration (less than 0.5 mM), the highest expression was detected in the GST fusion system with tac promoter, and the lowest level of expression appeared in the biotinylated fusion system. The expression level in the latter system did not differ dramatically with a range of different inducer concentrations. GST and 6× His fusion proteins were mainly soluble in aqueous solutions and Triton X-100 improved the solubility of biotinylated fusion proteins (inferring this protein is membrane-associated). Fusion proteins could only be partially purified by a single affinity chromatography step for all three systems. Glutathione-coupled matrix and streptavidin-conjugated resin have higher specificity than Ni–NTA resin, and elution conditions were shown to affect enzyme activity. Three kinds of recombinant 10-DABT with different tags showed enzyme activity, but total enzyme activity was lost as a result of the affinity chromatography step. Thrombin and Factor Xa could be used for site-specific cleavage of fusion proteins, but the incubation temperature affected enzyme activity of recombinant enzymes.

High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris

Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp) 4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK L) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK L by Mut s strain was much higher than that by Mut + strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK L (rEK L) reached 350 mg l −1, which was 20-fold higher than that reported previously. The recombinant EK L was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK L were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK L was approximately 9000 u mg −1. To facilitate purification and removal of rEK L after cleavage of fusion protein, the C-terminal His-tagged EK L (EK L/His) was also expressed in P. pastoris, and this His-tagged EK L exhibited a similar enzymatic activity to the untagged EK L.

Virokinin, a Bioactive Peptide of the Tachykinin Family, Is Released from the Fusion Protein of Bovine Respiratory Syncytial Virus

Tachykinins, an evolutionary conserved family of peptide hormones in both invertebrates and vertebrates, are produced by neuronal cells as inactive preprotachykinins that are post-translationally processed into different neuropeptides such as substance P, neurokinin A, and neurokinin B. We show here that furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein results in the release of a peptide that is converted into a biologically active tachykinin (virokinin) by additional post-translational modifications. An antibody directed to substance P cross-reacted with the C terminus of mature virokinin that contains a classical tachykinin motif. The cellular enzymes involved in the C-terminal maturation of virokinin were found to be present in many established cell lines. Virokinin is secreted by virus-infected cells and was found to act on the tachykinin receptor 1 (TACR1), leading to rapid desensitization of this G protein-coupled receptor as shown by TACR1-green fluorescent protein conjugate translocation from the cell surface to endosomes and by co-internalization of the receptor with beta-arrestin 1-green fluorescent protein conjugates. In vitro experiments with isolated circular muscle from guinea pig stomach indicated that virokinin is capable of inducing smooth muscle contraction by acting on the tachykinin receptor 3. Tachykinins and their cognate receptors are present in the mammalian respiratory tract, where they have potent effects on local inflammatory and immune processes. The viral tachykinin-like peptide represents a novel form of molecular mimicry, which may benefit the virus by affecting the host immune response.

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases.

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

Platelet-derived Growth Factor Induces the β-γ-Secretase-mediated Cleavage of Alzheimer's Amyloid Precursor Protein through a Src-Rac-dependent Pathway

The beta-amyloid peptide (Abeta) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as beta- and gamma-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APP-Gal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the gamma-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases Abeta generation and that, in cells stably expressing APP, the generation of A is strongly decreased by the Src tyrosine kinase inhibitor PP2.

Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp) 4–Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx–EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K m of 0.17 mM, and K cat is 20.8 s −1 . From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.

Furin is involved in baculovirus envelope fusion protein activation.

The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30-41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1 and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominantly as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.

Linkers for improved cleavage of fusion proteins with an engineered α‐lytic protease

Linkers for improved cleavage of fusion proteins with an engineered α‐lytic protease

Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion.

Preparations of purified full-length fusion (F) protein of human respiratory syncytial virus (HRSV) expressed in recombinant vaccinia-F infected cells, or of an anchorless mutant (F(TM(-))) lacking the C-terminal 50 amino acids secreted from vaccinia-F(TM(-))-infected cells contain a minor polypeptide that is an intermediate product of proteolytic processing of the F protein precursor F0. N-terminal sequencing of the intermediate demonstrated that it is generated by cleavage at a furin-motif, residues 106-109 of the F sequence. By contrast, the F1 N terminus derives from cleavage at residue 137 of F0 which is also C-terminal to a furin recognition site at residues 131-136. Site-directed mutagenesis indicates that processing of F0 protein involves independent cleavage at both sites. Both cleavages are required for the F protein to be active in membrane fusion as judged by syncytia formation, and they allow changes in F structure from cone- to lollipop-shaped spikes and the formation of rosettes by anchorless F.

Linkers for improved cleavage of fusion proteins with an engineered α‐lytic protease

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.

The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0(EHs)) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0(EHr)). The size and glycosylation state of F0(EHr) were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0(EHr) was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0(EHr) was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.

Accurate disulfide formation in Escherichia coli: overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI.

The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.

Design of high-throughput methods of protein production for structural biology

I greatly appreciate interacting with Peter Schultz, Scott Lesley and Marc Nasoff at the Genomics Institute for the Novartis Research Foundation and Mark Knuth and Ron Swanson at Syrrx, Inc., where much of this work is currently being pursued. I also thank Jim Graziano, Chris Lee, Mike Hanson and Marianne Patch for helpful suggestions on this manuscript.