Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation.

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.

Non-chromatographic purification of thermostable endoglucanase from Thermotoga maritima by fusion with a hydrophobic elastin-like polypeptide.

Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.

Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth.

Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with ß-glucosidase (Glu) to construct a recombinant Glu-linker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzyme-Cu3(PO4)2 nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a "binder" to assemble Cu3(PO4)2 nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu.

Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications.

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Designing an ELP-intein system: toward a more realistic outlook.

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.

Biotechnological applications of elastin-like polypeptides and the inverse transition cycle in the pharmaceutical industry.

Proteins are essential throughout the biological and biomedical sciences and the purification strategies of proteins of interest have advanced over centuries. Elastin-like polypeptides (ELPs) are compound polymers that have recently been highlighted for their sharp and reversible phase transition property when heated above their lower critical solution temperature (LCST). ELPs preserve this behavior when fused to a protein, and as a result providing a simple method to isolate a recombinant ELP fusion protein from cell contaminants by taking the solution through the soluble and insoluble phase of the ELP fusion protein, a technique designated as the inverse transition cycle (ITC). ITC is considered an inexpensive and efficient way of purifying recombinant ELP fusion proteins. In addition, ELPs render recombinant fusion protein more stability and a longer clear time in blood stream, which give ELPs a lot of valuable applications in the biotechnological and pharmaceutical industry. This article reviews the modernizations of ELPs and briefly highlights on the possible use of technologies such as the automatic piston discharge (APD) centrifuges to improve the efficiency of the ITC in the pharmaceutical industry to obtain benefits.

Protective Effect of Bovine Elastin Peptides against Photoaging in Mice and Identification of Novel Antiphotoaging Peptides.

This study aimed to investigate the protective effects of bovine elastin hydrolysates on UV-induced skin photoaging in mice and to identify the potent antiphotoaging peptides. Results showed that the ingestion of elastin peptides could obviously ameliorate epidermis hyperplasia and fibroblast apoptosis, and increase the content of hydroxyproline and water in photoaging skin in vivo ( p < 0.05). Furthermore, four peptides with elastase inhibitory activity were purified and identified, including GLPY, PY, GLGPGVG, and GPGGVGAL. Interestingly, GLPY and GPGGVGAL exhibited the highest inhibition activity with 58.77% and 42.91% at 10 mΜ, respectively. This might be attributed to the N-terminal Gly, C-terminal Leu, and Pro at the third position of the N-terminus, which showed stronger affinity and interaction with elastase. Moreover, GLPY and GPGGVGAL could also inhibit the apoptosis of fibroblasts effectively at 50 µΜ ( p < 0.01). It suggested that elastin peptides had great potential to prevent and regulate skin photoaging.

Elastin purification and solubilization.

The functional form of elastin is a highly cross-linked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of the mature protein is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove nonelastin "contaminates" are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis require careful attention during purification and in tropoelastin-based assays. This chapter describes the most common approaches for purification of elastin and for preparing solubilized forms of the protein.

Biocompatibility and immunogenicity of elastin-like recombinamer biomaterials in mouse models.

Novel thermo-sensitive elastin-like recombinamers (ELRs) containing bioactive molecules were created for use as a biomimetic biomaterial for tissue regeneration. For effective use for in vivo applications, it is essential to ensure that they do not induce adverse inflammatory, immune, or allergic responses that inhibit tissue repair. Therefore, we sought to establish a pre-clinical approach to evaluate biocompatibility in experimental mice using ELRs as a prototype biomaterial. First, we measured in vitro proliferation and cytokine production from BALB/c and C57BL/6 mouse splenocytes incubated with ELRs. Second, we used a rapid, high throughput in vivo approach in which inflammatory cells and cytokines were measured following an intraperitoneal implantation. Lastly, a subchronic in vivo approach was used in which ELRs or positive controls were subcutaneously implanted and the implantation sites were assessed for inflammation and gene expression. We found that ELRs induced mild inflammation and minimal fibrosis compared to the intense response to Vitoss. Additionally, implantation increased antigen-specific antibody titers for both groups and gene expression profiling of the implantation sites revealed the upregulation of inflammation, fibrosis, and wound healing-related genes in ELR and positive control-implanted mice compared to sham controls. These data demonstrate that ELRs appear safe for use in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 924-934, 2018.

Thermo-responsive human α-elastin self-assembled nanoparticles for protein delivery.

Self-assembled nanoparticles based on PEGylated human α-elastin were prepared as a potential vehicle for sustained protein delivery. The α-elastin was extracted from human adipose tissue and modified with methoxypolyethyleneglycol (mPEG) to control particle size and enhance the colloidal stability. The PEGylated human α-elastin showed sol-to-particle transition with a lower critical solution temperature (LCST) of 25°C-40°C in aqueous media. The PEGylated human α-elastin nanoparticles (PhENPs) showed a narrow size distribution with an average diameter of 330±33nm and were able to encapsulate significant amounts of insulin and bovine serum albumin (BSA) upon simple mixing at low temperature in water and subsequent heating to physiological temperature. The release profiles of insulin and BSA showed sustained release for 72h. Overall, the thermo-responsive self-assembled PhENPs provide a useful tool for a range of protein delivery and tissue engineering applications.

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

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.

Proliferative activity of elastin-like-peptides depends on charge and phase transition.

Elastin-like-peptides (ELPs) are stimulus-responsive protein-based polymers and are attractive biomaterials due to their biocompatibility and unique properties. This study shows that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 697-706, 2016.

Fibrous proteins: At the crossroads of genetic engineering and biotechnological applications.

Fibrous proteins, such as silk, elastin and collagen are finding broad impact in biomaterial systems for a range of biomedical and industrial applications. Some of the key advantages of biosynthetic fibrous proteins compared to synthetic polymers include the tailorability of sequence, protein size, degradation pattern, and mechanical properties. Recombinant DNA production and precise control over genetic sequence of these proteins allows expansion and fine tuning of material properties to meet the needs for specific applications. We review current approaches in the design, cloning, and expression of fibrous proteins, with a focus on strategies utilized to meet the challenges of repetitive fibrous protein production. We discuss recent advances in understanding the fundamental basis of structure-function relationships and the designs that foster fibrous protein self-assembly towards predictable architectures and properties for a range of applications. We highlight the potential of functionalization through genetic engineering to design fibrous protein systems for biotechnological and biomedical applications.

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

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass.

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported.

Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants.

Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins.

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human ß defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

The effect of glycation on arterial microstructure and mechanical response.

Like engineered materials, an artery's biomechanical behavior and function depend on its microstructure. Glycation is associated with both normal aging and diabetes and has been shown to increase arterial stiffness. In this study we examined the direct effect of glycation on the mechanical response of intact arteries and on the mechanical response and structure of elastin isolated from the arteries. Samples of intact arteries and isolated elastin were prepared from porcine aortas and glycated. The mechanical response of all samples was completed using a uniaxial material test system. Glycation levels were measured using ELISA. A confocal microscope was used to image differences in the structure of the glycated and untreated elastin fibers. We found that, under the conditions used in this study, glycation led to decreased stiffness of elastin isolated from arteries, which was associated with a thinning of elastin fibers as imaged by confocal microscopy. We observed no effect of glycation on collagen fibers under our treatment conditions. These results suggest that glycation leads to weakening of the elastin component of arteries that could contribute to vascular defects seen in diabetes and aging. Prevention of glycation reactions may be an important consideration for vascular health later in life.

Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification.