High yield expression of recombinant pro-resilin: Lactose-induced fermentation in E. coli and facile purification

Kim, Misook; Elvin, Christopher M.; Brownlee, A.; Lyons, Russell E.

doi:10.1016/j.pep.2006.11.003

Cited by 72 publications

(71 citation statements)

References 18 publications

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“…As with other RLP proteins, heating the cell-free lysate to 80–90°C selectively precipitated contaminating bacterial proteins from solution 12 and, when performed prior to Ni-affinity chromatography, helped enhance the purity of the final product. The inclusion of β-mercaptoethanol (10–20 mM) in the lysis and wash buffers helped prevent disulfide formation with contaminating bacterial proteins, but it left an unreactive adduct that had to be removed following purification.…”

Section: Resultsmentioning

confidence: 99%

“…The cleared lysate was then heated to 90°C for ten minutes and centrifuged again to remove precipitated protein. The heat stability and solubility of resilin-like polypeptides allows for the selective removal of contaminating bacterial proteins via a heating/precipitation procedure 12 and it improved the final purity of the RLP. 17 The cleared lysate was then purified under native conditions (10 mM β-mercaptoethanol) using Ni- NTA affinity chromatography and the purified protein was dialyzed against DI water to remove salts prior to lyophilization.…”

Section: Methodsmentioning

confidence: 99%

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Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

McGann¹,

Akins

Kiick

2015

Biomacromolecules

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Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility, and expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multi-arm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multi-arm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and crosslinking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, micro-heterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

McGann¹,

Akins

Kiick

2015

Biomacromolecules

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“…Cells were cultured at 37°C until an A 600 of 0.6 was reached, incubated for 5 min at 4°C, then induced for 3 h at 20°C with 0.1 mM isopropyl ␤-D-1-thiogalactopyranoside. Recombinant expression was according to Thomas and Baneyx (24) for RcPPC4 and Kim et al (25) for AtPPC3. The cultures were centrifuged and pellets were quick frozen in liquid N 2 and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants

O’Leary¹,

Rao²,

Kim³

et al. 2009

Journal of Biological Chemistry

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Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterialtype PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high K m(PEP) , and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC V max , particularly in the presence of the inhibitor L-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs. Phosphoenolpyruvate (PEP)2 carboxylase (PEPC; EC 4.1.1.31) is an important enzyme of plant C metabolism that catalyzes the irreversible ␤-carboxylation of phosphoenolpyruvate to yield oxaloacetate and P i . This enzyme has been intensively studied with regards to its crucial role in catalyzing atmospheric CO 2 fixation in C 4 and crassulacean acid metabolism photosynthesis (1, 2). It also plays essential functions in bacteria and non-green plant cells, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates withdrawn for biosynthesis and N-assimilation (3, 4). Most vascular plant PEPCs exist as a homotetrameric "dimer of dimer" structure composed of four identical 100 -110-kDa subunits known as Class-1 PEPC. Class-1 PEPCs are subject to tight control by a combination of allosteric effectors and reversible phosphorylation at a conserved N-terminal seryl residue catalyzed by a dedicated Ca 2ϩ -independent PEPC protein kinase and protein phosphatase type 2A (1-3). Allosteric activation by hexose phosphates and inhibition by malate have been routinely observed, whereas phosphorylation activates the enzyme by reducing its sensitivity to malate inhibition and simultaneously enhancing activation by hexose phosphates (1-3). Our understanding of the post-translational control...

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“…[21] Furthermore, high yield recombinant synthesis of resilin-like polypeptide has been reported, containing multiple copies of a repeat sequence within the resilin-like gene of D. melanogaster and Anopheles gambiae (African malaria mosquito). [22,23] The presence of repeated sequences in common with many other elastomeric proteins, prompted us to investigate the molecular structure of this protein by the use of model polypeptides based upon two different D. melanogaster resilin-repeat sequences. This approach has been successfully applied to elastin, abductin, lamprin, flagelliform silk and other elastomeric proteins, and used to identify multiconformational equilibria among poly-l-proline II (PPII), b-strands and b-turn conformations.…”

Section: Introductionmentioning

confidence: 99%

Molecular and Supramolecular Structural Studies on Significant Repetitive Sequences of Resilin

et al. 2009

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Resilin is a member of the family of elastomeric proteins and is found in specialised regions of the cuticle of most insects, and provides low stiffness, high strain and efficient energy storage. It is best known for its role in insect flight and the remarkable jumping ability of fleas and spittle bugs. In common with other elastomeric proteins, the recently identified Drosophila melanogaster proresilin shows glycine-rich repetitive sequences; in particular the N- and C-terminal regions of the protein are dominated by 18 repeats of a 15-residue sequence (SDTYGAPGGGNGGRP) and eleven repeats of a 13-residue sequence (GYSGGRPGGQDLG), respectively. We synthesised and analysed the molecular and supramolecular structure of some polypeptides with sequences belonging to the glycine-rich repeated domain of D. melanogaster resilin. The conformational studies performed by CD, FTIR and NMR spectroscopies pointed to the coexistence of two main conformational features, such as folded beta-turns and (quasi)extended structures (e.g., poly-L-proline II conformation) in common with other elastomeric proteins; this suggests an elasticity mechanism for resilin common to other elastomeric proteins. Our data show that also in the case of resilin, repetitive sequences are characterised by autonomous structures almost independent of the remaining parts of the molecule as already extensively found for elastin. From a supramolecular point of view, a great tendency to aggregate in fibrous structures is observed, particularly for the resilin- inspired polypeptide (PGGGN)(10). This is encouraging for the development of resilin-based biomaterials for the production of biocompatible medical devices, as well as high performing elastic materials.

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High yield expression of recombinant pro-resilin: Lactose-induced fermentation in E. coli and facile purification

Cited by 72 publications

References 18 publications

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants

Molecular and Supramolecular Structural Studies on Significant Repetitive Sequences of Resilin

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