Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Élisabeth

doi:10.1016/j.pep.2015.03.013

Cited by 17 publications

(12 citation statements)

References 35 publications

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“…While this value varied depending on the specific length of the construct, observed yields ranged 250660 mg/L. These results are a significant improvement over our in-house attempts to apply conventional ITC-only purification methods to this class of ELPs as well as previously reported yields of comparable marginally soluble, short ELPs that produced roughly 10 and 35 mg per liter of cells, respectively (12,13). The first purification step serves to remove a large number of contaminating proteins that would otherwise be carried over into the subsequent ITC step.…”

Section: Resultsmentioning

confidence: 56%

“…Some work has been carried out on shorter, hydrophobic ELPs using maltose binding protein (MBP) as a solubility and affinity-purification partner as a way to circumvent the above issues (12). This has, however, introduced another complication in that the MBP must then be separated from the hydrophobic ELP, an approach with varying levels of success depending on the ELP construct in question (12).…”

mentioning

confidence: 99%

“…This has, however, introduced another complication in that the MBP must then be separated from the hydrophobic ELP, an approach with varying levels of success depending on the ELP construct in question (12). More recently an ITC-only purification approach has been used to produce the ELPs without the interfering MBP, but the yields of purified protein severely limit the applications of such an approach (13).…”

mentioning

confidence: 99%

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High-Yield Recombinant Expression and Purification of Marginally Soluble, Short Elastin-Like Polypeptides

2016

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The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

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Section: Resultsmentioning

confidence: 56%

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confidence: 99%

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confidence: 99%

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High-Yield Recombinant Expression and Purification of Marginally Soluble, Short Elastin-Like Polypeptides

2016

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“…ELP or tropoelastin production has been described in E. coli, [117] a number of plant species [118] and yeast. [119] Electrospun silk proteins have also attracted increasing interest as biopolymers in recent years due to the extreme strength of natural fibers such as spider dragline silk.…”

Section: Expression Systems For Recombinant Productsmentioning

confidence: 99%

Adding Functions to Biomaterial Surfaces through Protein Incorporation

et al. 2016

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The concept of biomaterials has evolved from one of inert mechanical supports with a long-term, biologically inactive role in the body into complex matrices that exhibit selective cell binding, promote proliferation and matrix production, and may ultimately become replaced by newly generated tissues in vivo. Functionalization of material surfaces with biomolecules is critical to their ability to evade immunorecognition, interact productively with surrounding tissues and extracellular matrix, and avoid bacterial colonization. Antibody molecules and their derived fragments are commonly immobilized on materials to mediate coating with specific cell types in fields such as stent endothelialization and drug delivery. The incorporation of growth factors into biomaterials has found application in promoting and accelerating bone formation in osteogenerative and related applications. Peptides and extracellular matrix proteins can impart biomolecule- and cell-specificities to materials while antimicrobial peptides have found roles in preventing biofilm formation on devices and implants. In this progress report, we detail developments in the use of diverse proteins and peptides to modify the surfaces of hard biomaterials in vivo and in vitro. Chemical approaches to immobilizing active biomolecules are presented, as well as platform technologies for isolation or generation of natural or synthetic molecules suitable for biomaterial functionalization.

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“…Widely used tags include: hexahistidine (His-tag), the most popular affinity tag for the isolation and purification of proteins via immobilized metal ion affinity chromatography (IMAC) [15,16]; maltose-binding protein (MBP) tags, which bind to amylose [18]; glutathione-S-transferase (GST) tags, which fuse to the proteins of interest and are purified using immobilized glutathione columns [19]; and Strep-tag II [20].…”

Section: Introductionmentioning

confidence: 99%

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

Han

Ming

Deng

et al. 2017

Biochemical and Biophysical Research Communications

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Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

Cited by 17 publications

References 35 publications

High-Yield Recombinant Expression and Purification of Marginally Soluble, Short Elastin-Like Polypeptides

High-Yield Recombinant Expression and Purification of Marginally Soluble, Short Elastin-Like Polypeptides

Adding Functions to Biomaterial Surfaces through Protein Incorporation

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

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