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Abstract Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli , resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2–12), temperature (20–50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure. Highlights • The full length dHC-serpin gene cloned from Hyphantria cunea Drury. • The SUMO-dHC-serpin fusion protein is highly soluble in Escherichia coli. • dHC-serpin exhibits activity against papain. • dHC-serpin remained active and stable between 20 °C and 50 °C and pH 2 and 12. • The reactive center loop region is important for dHC-serpin binding with papain. [ABSTRACT FROM AUTHOR]

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris , we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The Sac I-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4–20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein. [ABSTRACT FROM AUTHOR]

A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination. [ABSTRACT FROM AUTHOR]

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli . To improve the expression level of ABP-dHC-cecropin A in E. coli , tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors ( n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins ( n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A. [ABSTRACT FROM AUTHOR]

The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays a role in facilitating the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens and is also involved in MHC I-restricted antigens in adaptive immunity catalyzing disulfide bond reduction in mammals. In this study, we cloned a GILT gene homolog from largemouth bass (designated ‘lbGILT’), a freshwater fish belonging to Perciformes and known for its nutritive value. We obtained the full-length cDNA of lbGILT by reverse transcription PCR and rapid amplification of cDNA ends. This cDNA is comprised of a 5'-untranslated region (UTR) of 87 bp, a 3′-UTR of 189 bp, and an open reading frame of 771 bp. It encodes a protein of 256 amino acids with a deduced molecular weight of 28.548 kDa and a predicted isoelectric point of 5.62. The deduced protein possesses the typical structural features of known GILTs, including an active site motif, two potential N-linked glycosylation sites, a GILT signature sequence, and six conserved cysteines. Tissue-specific expression of lbGILT was shown by real-time quantitative PCR. The expression of lbGILT mRNA was obviously up regulated in spleen and kidney after induction with lipopolysaccharide. Recombinant lbGILT was produced as an inclusion body with a His 6 tag in ArcticExpress (DE3), and the protein was then washed, solubilized, and refolded. The refolded lbGILT showed reduction activity against an IgG substrate. These results suggest that lbGILT plays a role in innate immunity. [ABSTRACT FROM AUTHOR]