High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

Zerfaß, Christian; Braukmann, Sandra; Nietzsche, Sándor; Hobe, Stephan; Paulsen, Harald

doi:10.1016/j.pep.2014.12.012

Cited by 11 publications

(26 citation statements)

References 66 publications

(49 reference statements)

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“…In addition, as mineralizing peptides or proteins normally have high‐cationic charge density, their strong interactions with anionic cellular compounds such as nucleic acids (Olins et al, ) or membrane lipopolysaccharides (Rietschel et al, ) under neutral pH render their recovery and isolation challenging. These problems are usually addressed by the fusion of the mineralizing biomolecules to carrier proteins, for example, poly(histidine) (Sano et al, ; Tahir et al, ; Wong Po Foo et al, ; Zhou et al, ), maltose‐binding protein (Zhou et al, ), and ketosteroid isomerase (Zerfaß et al, ) tags, to facilitate downstream processing reliant mainly on chromatography. The fusion tags can either be subsequently cleaved with further purification or be left combined with the target biomolecules as long as retention does not compromise intended functionality.…”

Section: Introductionmentioning

confidence: 99%

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Wibowo

Yang

Middelberg

et al. 2016

Biotech & Bioengineering

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Inspired by nature, synthetic mineralizing proteins have been developed to synthesize various structures of silica-based nanomaterials under environmentally friendly conditions. However, the development of bioprocesses able to assist in the translation of these new materials has lagged the development of the materials themselves. The development of cost-effective and scalable bioprocesses which minimize reliance on chromatography to recover biomolecules from microbial cell factories remains a significant challenge. This paper reports a simplified purification process for a recently reported recombinant catalytic modular (D4S2) protein (M(DPSMKQLADS-LHQLARQ-VSRLEHA) EPSRKKRKKRKKRKKGGGY; M 13.3 kDa; pI 10.9), which combines a variant of the established designer biosurfactant protein DAMP4 with a new biomimetic sequence (RKKRKKRKKRKKGGGY), providing for a bi-modular functionality (emulsification and biosilicification). The four-helix bundle structure of the protein has been demonstrated to remain stable and soluble under high temperature and high salt conditions, which confers simplified bioprocessing character. However, the high positive charge on the biosilification sequence necessitates removal of DNA contaminants from crude cell-extract at an early stage in the process by adding poly(ethyleneimine) (PEI). In this process, cellular protein contaminants were selectively precipitated by adding Na SO to the protein mixture up to a high concentration (1 M) and mixed at high temperature (90°C, 5 min) where D4S2 remained stable and soluble due to its four-helix bundle structure. Further increase of the Na SO concentration to 1.8 M precipitated, thus separated, D4S2 from residual PEI. The overall yield of the protein D4S2 was 28.8 mg per 800 mL cells (final cultivation OD ∼2) which gives an approximate 79% D4S2-protein yield. In comparison with the previously reported chromatographic purification of D4S2 protein (Wibowo et al., 2015), the final yield of D4S2 protein is increased fourfold in this study. The bio-produced protein D4S2 was proved to retain it emulsification and biosilicification functionalities enabling the formation of oil-core silica-shell nanocapsules at near-neutral pH and room temperature without the use of any toxic organic solvents, confirming no adverse effects due to bioprocess simplification. This work demonstrates that, through proper bioprocess engineering including the removal of critical contaminants such as DNA, a more efficient, simple, and scalable purification process can be used for the high-yield bio-production of a recombinant templating protein useful in the synthesis of bio-inspired nanomaterials. This simplified process is expected to be easily adapted to recover other mineralizing helix bundle-based functional proteins from microbial cell factories. Biotechnol. Bioeng. 2017;114: 335-343. © 2016 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Wibowo

Yang

Middelberg

et al. 2016

Biotech & Bioengineering

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“…Full details of the expression and purification of recombinant P 5 S 3 in Escherichia coli have been reported earlier . In brief, the peptide was expressed as a fusion protein with ketosteroid isomerase (KSI), which mediates the deposition of the expressed protein in inclusion bodies .…”

Section: Methodsmentioning

confidence: 99%

“…The R5 and PL12 peptides are partial sequences of Cylindrotheca fusiformis Sil1 2 and Thalassiosira pseudonana Sil3, respectively. Conversely, P 5 S 3 is a tailored peptide that can be produced by recombinant bacterial expression and is active in silicic‐acid mineralization and stabilization . Basic amino acids are highlighted in blue (underlined) [Color figure can be viewed at http://wileyonlinelibrary.com]…”

Section: Introductionmentioning

confidence: 99%

“…The impact of protein phosphorylation on the interaction with silica or silicic acid was studied by making use of multiple kinase target sites introduced into the P 5 S 3 sequence. In solutions of highly supersaturated silicic acid (that is, 30 mM, 15‐ to 30‐fold supersaturated), P 5 S 3 , both in its phosphorylated and non‐phosphorylated states, accelerated the formation of silica between pH 6.5 and 9.5 . At less supersaturated concentrations of silicic acid (that is, 8.3 mM, 4‐ to 8‐fold supersaturated) the peptide impeded the silicic acid polymerization to silica.…”

Section: Introductionmentioning

confidence: 99%

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Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P₅S₃ during silicic acid condensation and silica decondensation

et al. 2017

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The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P5S3, a 50-residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains five protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P5S3 enhances silica-formation whereas it retards the dissolution of amorphous silica (SiO2) at globally undersaturated concentrations. The secondary structure of P5S3 during these two processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in the absence of silicate. The NMR studies of dual-labeled (13C, 15N) P5S3 revealed a disordered structure at pH 2.8 and 4.5. Within the pH range of 4.5 to 9.5 in the absence of silicic acid, the CD data showed a disordered structure with the suggestion of some polyproline II character. Upon silicic acid polymerization and during dissolution of preformed silica, the CD spectrum of P5S3 indicated partial transition into an α-helical conformation which was transient during silica-dissolution. The secondary structural changes observed for P5S3 correlate with the presence of oligomeric/polymeric silicic acid, presumably due to P5S3-silica interactions. These P5S3-silica interactions appear, at least in part, ionic in nature since negatively charged dodecylsulfate caused similar perturbations to the P5S3 CD spectrum as observed with silica, while uncharged ß-D-dodecyl maltoside did not affect the CD spectrum of P5S3. Thus, with an associated increase in α-helical character, P5S3 influences both the condensation of silicic acid into silica and its decondensation back to silicic acid.

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“…After induction with isopropyl-beta-D-thiogalactopyranoside, cells were harvested and then lysed in Tris-HCl buffer (6 M guanidine, pH 7.9). A HisPur Ni-NTA resin kit (Thermo Scientific) was used for the isolation of the fusion protein, followed by cyanogen bromide cleavage (Zerfaß et al, 2014). After the removal of precipitated ketosteroid isomerase, the ALW peptide was lyophilized.…”

Section: Methodsmentioning

confidence: 99%

Anti-DNA antibody mediated catalysis is isotype dependent

Xia

Eryilmaz

Zhang

et al. 2016

Molecular Immunology

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Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.

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High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

Cited by 11 publications

References 66 publications

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P₅S₃ during silicic acid condensation and silica decondensation

Anti-DNA antibody mediated catalysis is isotype dependent

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High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

Cited by 11 publications

References 66 publications

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P5S3 during silicic acid condensation and silica decondensation

Anti-DNA antibody mediated catalysis is isotype dependent

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Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P₅S₃ during silicic acid condensation and silica decondensation