Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels

A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC₅₀ value of 156.16 nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.     

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern

Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (-C-C-C-C-) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to alpha-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.     

A novel M-superfamily conotoxin with a unique motif from Conus vexillum

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds for the purpose of feeding and defence, each evolved to act in a highly specific manner on different parts of the nervous system. Here, we report the peptide purification, molecular cloning, chemical synthesis, and functional characterization of a structurally unique toxin isolated from the venom of Conus vexillum. The novel peptide, designated Vx2, was composed of 21 amino acid residues cross-linked by 3 disulfide bonds (WIDPSHYCCCGGGCTDDCVNC). Intriguingly, its mature peptide sequence shows low level of similarity with other identified conotoxins, and its unique motif (-CCCGGGC-) was not reported in other Conus peptides. However, its signal peptide sequence shares high similarity with those of the M-superfamily conotoxins. Hence, Vx2 could be classified into a new family of the M-superfamily.     

Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus

A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.     

A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.     

Soluble expression and purification of the functional interleukin-30 protein in Escherichia coli

Interleukin-30 (IL-30), or IL-27p28, is the α subunit of IL-27 constructed by Epstein–Barr virus-induced gene 3 (EBI3) and IL-27p28 binding via noncovalent bonds. IL-30 can be independently secreted and function independently of IL-27. Recent studies demonstrated IL-30 could concurrently antagonize T helper 1 (Th1) and Th17 responses and might have therapeutic implications for controlling autoimmune diseases. However, no reports have stated an efficient method to generate a relatively large quantity of IL-30. In this study, an Escherichia coli expression system for the rapid expression of the mouse IL-30 is developed. For the first time, IL-30 was expressed in a form of soluble fusion protein and purified using a method of simple affinity chromatography. In order to avoid the impact of minor codons on expressing eukaryotic protein in E. coli and to improve the expression quantity, the nucleotide sequence of IL-30 was optimized. The optimized gene sequence was then subcloned into the pET-44a(+) vector, which allowed expression of IL-30 with a fusion tag, NusA. The vector was transformed into E. coli and the expressed fusion protein, NusA-IL-30, was purified by Ni chromatography. Then the fusion tag was removed by cleavage with thrombin. The purity of purified IL-30 was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as high-performance liquid chromatography (HPLC) and the purity was up to about 92%. The yield of IL-30 was 8.95 mg from 1 L of bacterial culture. Western blot confirmed the identity of the purified protein. The recombinant IL-30 showed its biological activity by inhibiting Th17 differentiating from naive CD4⁺ T cells. Therefore, this method of express and purifying IL-30 provides novel procedures to facilitate structural and functions studies of IL-30.     

Soluble expression and purification of porcine pepsinogen from Pichia pastoris

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).     

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.     

A novel conotoxin from Conus betulinus,kappa-BtX,unique in cysteine pattern and in function as a specific BK channel modulator

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.     

Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion

Cyanovirin-N (CVN) is a promising antiviral candidate that has an extremely low sequence homology with any other known proteins. The efficient and soluble expression of biologically functional recombinant CVN (rCVN) is still an obstacle due to insufficient yield, aggregation, and abnormal modification. Here, we describe an improved approach to preparing native rCVN from Escherichia coli more efficiently. A fusion gene consisting of cvn and sumo (small ubiquitin-related modifier) and a hexahistidine tag was constructed according to the codon bias of the host cell. This small ubiquitin-related modifier (SUMO)-fused CVN is expressed in the cytoplasm of E. coli in a folded and soluble form (>30% of the total soluble protein), yielding 3 to 4 mg of native rCVN from 1 g of wet cells to a purity up to 97.6%. Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry and reverse-phase high-performance liquid chromatographic analysis showed that the purified rCVN was an intact and homogeneous protein with a molecular weight of 11,016.68 Da. Potent antiviral activity of rCVN against herpes simplex virus type 1 and human immunodeficiency virus type 1/IIIB was confirmed in a dose-dependent manner at nanomolar concentrations. Thus, the His-SUMO double-fused CVN provides an efficient approach for the soluble expression of rCVN in the cytoplasm of E. coli, allowing an alternative system to develop bioprocess for the large-scale production of this antiviral candidate.     

A novel sodium channel inhibitor from Conus geographus: purification, structure, and pharmacological properties

A novel toxin, tentatively named conotoxin GS (CGS), has been isolated from a marine snail, Conus geographus. CGS was found to exist as a single polypeptide chain, consisting of 34 amino acid residues, cross-linked by three disulfide bonds. Its amino acid sequence was shown to be Ala-Cys-Ser-Gly-Arg-Gly-Ser-Arg-Cys-Hyp-Hyp-Gln-Cys-Cys-Met-Gly-Leu-Arg- Cys-Gly - Arg-Gly-Asn-Pro-Gln-Lys-Cys-Ile-Gly-Ala-His-Gla-Asp-Val. In competition experiments, CGS inhibited the bindings of [3H]Lys-tetrodotoxin ([3H]Lys-TTX) and [3H]propionylconotoxin GIIIA to Electrophorus electricus electroplax membranes, with Ki values of 34 nM and 24 nM, respectively. The toxin inhibited the binding of [3H]Lys-TTX (1 nM) to rat skeletal muscle homogenates with an IC50 value of 880 nM but showed very little effect on this binding to the rat brain P2 fraction at 10 microM. These binding studies indicate that CGS belongs to the same group of Na channel inhibitors as TTX, STX (saxitoxin), and mu-conotoxins. Although CGS, like the mu-conotoxins, is a pharmacological probe for distinguishing between neuronal and muscle Na channel subtypes, the homology in the sequences of CGS and mu-conotoxins is very limited.     

Molecular cloning, functional expression and purification of a glucan branching enzyme from Neisseria denitrificans(1).

The nucleotide sequence containing the complete structural information for a glucan branching enzyme was isolated from a Neisseria denitrificans genomic library. The gene was expressed in Escherichia coli and the active recombinant protein was purified. The deduced protein of 762 amino acids with a calculated molecular weight of 86313 Da shows similarity to the primary protein sequences of other known glucan branching enzymes. Amino acid sequencing of the isolated protein by Edman degradation confirmed the deduced start codon of the structural gene of the glucan branching enzyme. The purified glucan branching enzyme has a stimulating effect on the Neisseria amylosucrase activity.     

Prokaryotic expression,purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42°C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.     

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.     

Soluble expression and one-step purification of a neurotoxin Huwentoxin-I in Escherichia coli

Huwentoxin-I (HWTX-I) is a small 33-amino acid neurotoxin from the venom of the Chinese bird spider Ornithoctonus huwena. HWTX-I selectively blocks N-type voltage-sensitive calcium channels (N-VSCCs) and has great potential for clinical application as a novel analgesic without inducing drug tolerance. However, there are still many unsolved issues for this peptide, such as its clinical efficacy in analgesia, anesthesia, and even its potential role in drug rehabilitation. Therefore, large amounts of active recombinant HWTX-I are urgently needed. In this report, we describe a novel and efficient way to produce large amounts of the valuable form in Escherichia coli. HWTX-I was expressed in soluble form as an N-terminal intein fusion product. After affinity purification, a pH shift-induced self-cleavage of the intein released HWTX-I, resulting in a single-column purification of the target protein. The whole-cell patch clamp assay showed that purified HWTX-I has activity similar to another commercialized N-VSCC blocker ω-conotoxin MVIIA. Production of HWTX-I by this method has the major advantages of high efficiency and low cost.     

Isolation,structural identification and biological characterization of two conopeptides from the Conus pennaceus venom

A novel anti-mollusk conopeptide pn4c was isolated from the Conus pennaceus venom by repeated HPLC fractionation based on the activity against freshwater snails. The primary structure of pn4c was determined by the mass spectrometric de novo sequencing analysis. In addition, pn3a was isolated from the same fraction containing pn4c, as a peptide with unknown functions.     

Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.