To study the effect of physiological conditions on spidroins, we analyzed NTR1SR2CT module secondary structure, aggregation and silk-formation influenced by different salts (in different concentration intervals). According to the full-length Araneus ventricosus MiSp sequence, NTR1SR2CT module was constructed and expressed in Escherichia coli BL21 (DE3), and the recombinant proteins were purified by denaturation method mediated by 8 mol/L urea. Random coil and helix are the main secondary structures of NTR1SR2CT and could be induced into beta-sheet by drying natively and lyophilization, where methanol can be used as a promoter. Furthermore, potassium and phosphate cations can cause significant NTR1SR2CT protein aggregation and silk-formation. The results could be a basis for the determination of silk-formation mechanism, and also useful for industrialized generation of high performance spider silk-like fibers.
Animals ; Fibroins ; chemistry ; Protein Structure, Secondary ; Salts ; chemistry ; Spiders

Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.
Animals ; Arthropod Proteins ; chemistry ; Protein Engineering ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Spiders

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.
Animals ; Inteins ; Protein Splicing ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Small Ubiquitin-Related Modifier Proteins ; chemistry ; Spiders ; Trans-Splicing

Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Codon ; DNA, Complementary ; chemistry ; Fibroins ; Molecular Sequence Data ; Polymerase Chain Reaction ; Protein Structure, Secondary ; Proteins ; chemistry ; genetics ; Spiders

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.
Adenocarcinoma/*drug therapy/physiopathology ; Animals ; Apoptosis/drug effects ; Dog Diseases/*drug therapy/physiopathology ; Dogs ; Female ; Mammary Neoplasms, Animal/*drug therapy/physiopathology ; Mammary Neoplasms, Experimental/*drug therapy/physiopathology ; Mitosis/drug effects ; Spiders/*chemistry

RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.
Amino Acid Sequence ; Animals ; Base Sequence ; Chromatography, Affinity ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Fibroins ; genetics ; metabolism ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Plasmids ; genetics ; Recombinant Proteins ; chemistry ; isolation & purification ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid ; Spiders ; genetics ; metabolism

Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.
Amino Acid Substitution ; Animals ; Arginine ; genetics ; Ganglia, Spinal ; drug effects ; Mutagenesis, Site-Directed ; Mutant Proteins ; pharmacology ; Neurons ; drug effects ; Patch-Clamp Techniques ; Peptides ; chemistry ; genetics ; pharmacology ; Rats ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; drug effects ; Spider Venoms ; chemistry ; genetics ; isolation & purification ; pharmacology ; Spiders ; Tetrodotoxin ; pharmacology