Development of Self-Assembling Mixed Protein Micelles with Temperature-Modulated Avidities

Elastin-like polypeptides (ELPs) are polypentapeptides that undergo hydrophobic collapse and aggregation above a specific transition temperature, Tt. ELP diblocks sharing a common “core” block (I60) but varying “outer” blocks (A80, P40) were designed, where Tt,I \u3c Tt,A \u3c Tt,P. The formation of ~55 nm diameter mixed micelles from these ELP diblocks was verified using dynamic light scattering (DLS), multiangle light scattering (MALS) and fluorescence resonance energy transfer (FRET). To confer affinity to the blood circulating protein fibrinogen, a fibrinogen-binding tetrapeptide sequence (GPRP) was fused to A80-I60, while P40-I60 was fused to a non-binding control (GPSP). The self-assembling, peptide-displaying, mixed micelles exhibit temperature-modulated avidities for immobilized and soluble fibrinogen at 32 °C and 42 °C. In this initial proof-of-concept design, the engineered mixed micelles were shown to disengage fibrinogen at elevated temperatures. The modular nature of this system can be used for developing in vivo depot systems that will only be triggered to release in situ upon specific stimuli

Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium

Proteolytic enzyme engineering : a tool for wool

One of the goals of protein engineering is to tailor the structure of enzymes to optimize industrial bioprocesses. In the present work, we present the construction of a novel high molecular weight subtilisin, based on the fusion of the DNA sequences coding for Bacillus subtilis prosubtilisin E and for an elastin-like polymer (ELP). The resulting fusion protein was biologically produced in Escherichia coli, purified and used for wool finishing assays. When compared to the commercial protease Esperase, the recombinant subtilisinE-VPAVG220 activity was restricted to the cuticle of wool, allowing a significant reduction of pilling, weight loss and tensile strength loss of wool fibers. Here we report, for the first time, the microbial production of a functionalized high molecular weight protease for controlled enzymatic hydrolysis of wool surface. This original process overcomes the unrestrained diffusion and extended fiber damage which are the major obstacles for the use of proteases for wool finishing applications

Human adipose derived stem cells are superior to human osteoblasts (HOB) in bone tissue engineering on a collagen-fibroin-ELR blend

The ultrastructure of the bone provides a unique mechanical strength against compressive, torsional and tensional stresses. An elastin-like recombinamer (ELR) with a nucleation sequence for hydroxyapatite was incorporated into films prepared from a collagen – silk fibroin blend carrying microchannel patterns to stimulate anisotropic osteogenesis. SEM and fluorescence microscopy showed the alignment of adipose-derived stem cells (ADSCs) and the human osteoblasts (HOBs) on the ridges and in the grooves of microchannel patterned collagen-fibroin-ELR blend films. The Young's modulus and the ultimate tensile strength (UTS) of untreated films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28 days of cell culture, ADSC seeded film had a Young's modulus of 1.21 ± 0.42 MPa and UTS of 0.32 ± 0.15 MPa which were about 3 fold higher than HOB seeded films. The difference in Young's modulus was statistically significant (p: 0.02). ADSCs attached, proliferated and mineralized better than the HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissue engineering of collagen-fibroin-ELR based constructs used in this study. We have thus shown the enhancement in the tensile mechanical properties of the bone tissue engineered scaffolds by using ADSCs

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble–insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h–induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in ~75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins