Secreted production of self-assembling peptides in Pichia pastoris by fusion to an artificial highly hydrophilic protein

Moers, Antoine P. H. A.; Wolbert, E.J.H.; Wolf, Frits A. de; Werten, Marc W. T.

doi:10.1016/j.jbiotec.2010.01.010

Cited by 11 publications

(10 citation statements)

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“…Although aberrant migration of the proteins was expected, the presence of two bands suggests partial degradation. Because attachment of a short peptide with varied amino acid composition previously increased the migration rate of C P 2 in SDS–PAGE (Moers et al, ), the lower bands seen here may well correspond to the intact T 9 ‐C P 4 ‐D A and T 9 ‐C P 4 ‐D B proteins, and the upper bands may represent the proteins with most of the leucine zipper peptides missing owing to degradation. In agreement with this notion, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 74%

Production in Pichia pastoris of protein‐based polymers with small heterodimer‐forming blocks

Domeradzka

Werten

Vries

et al. 2015

Biotech & Bioengineering

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Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

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Section: Resultsmentioning

confidence: 74%

Production in Pichia pastoris of protein‐based polymers with small heterodimer‐forming blocks

Domeradzka

Werten

Vries

et al. 2015

Biotech & Bioengineering

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“…Although aberrant migration of the proteins was expected, the presence of two bands suggests partial degradation. Because attachment of a short peptide with varied amino acid composition previously increased the migration rate of C P 2 in SDS-PAGE (Moers et al, 2010), the lower bands seen here may well correspond to the intact T 9 -C P 4 -D A and T 9 -C P 4 -D B proteins, and the upper bands may represent the proteins with most of the leucine zipper peptides missing owing to degradation. In agreement with this notion, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 72%

“…Proteins were purified from the cell-free broth by differential ammonium sulfate precipitation, and subsequently dialyzed and lyophilized. Previous studies showed that ammonium sulfate precipitation of protein polymers containing the block typically results in a purity of ~99 % at the protein level [19, 30]. The gravimetrically determined yields, expressed in g per L of cell-free broth, were 2.2 g L −1 for , and 2.3 g L −1 for .…”

Section: Resultsmentioning

confidence: 98%

“…1). Based on the well-established aberrant migration behavior of the block in SDS-PAGE, the proteins were expected to migrate much more slowly in SDS-PAGE than would be expected on the basis of their theoretical molecular weights of ~41 and ~39 kDa, respectively [18, 19, 23, 30]. This is due to the highly hydrophilic character and consequent low SDS-binding capacity of the block [18].…”

Section: Resultsmentioning

confidence: 99%

“…The 13-residue D PPxY block resembles the proline-rich nature of the block, and as such is not expected to much affect the mobility of the protein in SDS-PAGE relative to that of alone. On the other hand, the 37-residue hydrophobic D WW block likely will, because attachment to of peptides that improve SDS binding is known to increase the mobility of the polymer [23, 30]. Indeed, the proteins migrated accordingly in SDS-PAGE (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains

et al. 2016

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BackgroundSpecific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing.ResultsWe constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P.pastoris with a purified protein yield of more than 2 g L−1 of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM.ConclusionsThis article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0498-3) contains supplementary material, which is available to authorized users.

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Antimicrobial Peptides

Marcos¹,

Manzanares²

2011

Antimicrobial Polymers

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Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of protegrin. Comparison of the modeling and experimental data suggests that approximately 10-100 protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis-a redundant ''overkill'' mechanism most likely to occur in locales with high protegrin concentrations. Although our study is limited to protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of protegrin.

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Secreted production of self-assembling peptides in Pichia pastoris by fusion to an artificial highly hydrophilic protein

Cited by 11 publications

References 41 publications

Production in Pichia pastoris of protein‐based polymers with small heterodimer‐forming blocks

Production in Pichia pastoris of protein‐based polymers with small heterodimer‐forming blocks

Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains

Antimicrobial Peptides

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