Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A.
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ABSTRACT:
SUBMITTER: Andersons D
PROVIDER: S-EPMC1130623 | biostudies-other | 1991 Nov
REPOSITORIES: biostudies-other
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Project description:Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.
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Project description:In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.
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Project description:Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation.
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Project description:Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.
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Project description:Four strains of the fungus Quambalaria cyanescens (Basidiomycota: Microstromatales), were used for the determination of secondary metabolites production and their antimicrobial and biological activities. A new naphthoquinone named quambalarine A, (S)-(+)-3-(5-ethyl-tetrahydrofuran-2-yliden)-5,7,8-trihydroxy-2-oxo-1,4-naphthoquinone (1), together with two known naphthoquinones, 3-hexanoyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone (named here as quambalarine B, 2) and mompain, 2,5,7,8-tetrahydroxy-1,4-naphthoquinone (3) were isolated. Their structures were determined by single-crystal X-ray diffraction crystallography, NMR and MS spectrometry. Quambalarine A (1) had a broad antifungal and antibacterial activity and is able inhibit growth of human pathogenic fungus Aspergillus fumigatus and fungi co-occurring with Q. cyanescens in bark beetle galleries including insect pathogenic species Beauveria bassiana. Quambalarine B (2) was active against several fungi and mompain mainly against bacteria. The biological activity against human-derived cell lines was selective towards mitochondria (2 and 3); after long-term incubation with 2, mitochondria were undetectable using a mitochondrial probe. A similar effect on mitochondria was observed also for environmental competitors of Q. cyanescens from the genus Geosmithia.
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