Purification of a Hydrophobic Elastin-Like Protein Toward Scale-Suitable Production of Biomaterials

Abstract

Elastin-like proteins (ELPs) are polypeptides with potential applications as renewable bio-based high-performance polymers, which undergo a stimulus-responsive reversible phase transition. The ELP investigated in this manuscript—ELP[V2Y-45]—promises fascinating mechanical properties in biomaterial applications. Purification process scalability and purification performance are important factors for the evaluation of potential industrial-scale production of ELPs. Salt-induced precipitation, inverse transition cycling (ITC), and immobilized metal ion affinity chromatography (IMAC) were assessed as purification protocols for a polyhistidine-tagged hydrophobic ELP showing low-temperature transition behavior. IMAC achieved a purity of 86% and the lowest nucleic acid contamination of all processes. Metal ion leakage did not propagate chemical modifications and could be successfully removed through size-exclusion chromatography. The simplest approach using a high-salt precipitation resulted in a 60% higher target molecule yield compared to both other approaches, with the drawback of a lower purity of 60% and higher nucleic acid contamination. An additional ITC purification led to the highest purity of 88% and high nucleic acid removal. However, expensive temperature-dependent centrifugation steps are required and aggregation effects even at low temperatures have to be considered for the investigated ELP. Therefore, ITC and IMAC are promising downstream processes for biomedical applications with scale-dependent economical costs to be considered, while salt-induced precipitation may be a fast and simple alternative for large-scale bio-based polymer production.

Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

“…Separation of the fusion protein was successful at 4 °C. Other fusion tags such as the ELP, hydrophobin and CspB are used for non-chromatographic purification under room temperature [6,32,33]. Different from the intein in the annexin fusion to potentially limit protein solubility [6], and the CSQ unable to be removed from the ZZ-CSQ [7], our constructs will be used for yielding the non-tagged target proteins via specific protease for cleaving the sequence introduced between the hanA1 and target protein, similar to the other proteases recognizing sequences incorporated between the ELP tag and target protein [34].…”

“…Removal of impurities during the precipitation/resolubilization process requires high speed centrifugation. For the ELP and hydrophobin tags, the expensive temperature-dependent centrifugation steps are required [32,33]. As a comparison, addition the inexpensive affinity resins, such as microcrystalline cellulose or RAC to the clear lysate for absorbing the CBM tagged protein can be separated from the unbound proteins by relatively low speed centrifugation at room temperature.…”

Mechanical Properties of Protein-Based Hydrogels Derived from Binary Protein Mixtures—A Feasibility Study

“…To maintain the hydrophobic ELP constructs in a disaggregated state at room temperature [ 32 ] and to allow comparable conditions for all samples, both buffer solutions contain 4 M urea, which causes (partial) protein unfolding for globular proteins and reduces casein micelle formation by disrupting intra- and intermolecular hydrophobic interactions [ 41 , 47 ]. Thus, two effects may occur due to the non-native protein structure, which may be additionally influenced by the different buffer components.…”

“…Following the nomenclature introduced by Meyer et al [ 31 ], the used hydrophobic ELP is referred to as ELP[V2Y-45] with the guest residues valine and tyrosine in a 2:1 ratio and with a total of 45 repetitions of the pentapeptide sequence. Fermentation and purification were performed using the inverse transition cycling (ITC) process previously described [ 32 ]. Briefly, the homogenized and subsequently centrifuged Escherichia coli lysate was resuspended in a buffer containing 4 M urea to dissolve inclusion bodies containing the ELP constructs.…”

“…If necessary, Vivaspin ® ultrafiltration units (Sartorius Stedim Biotech, Göttingen, Germany) with a molecular weight cut-off of 10 kDa were used according to the manufacturer’s recommendation to reach higher stock solution concentrations. Protein concentrations were determined with a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) using the extinction coefficients ε BSA,280 nm = 0.67 L/(g·cm) [ 33 ], ε α-Amylase,280 nm = 2.60 L/(g·cm) [ 34 ], ε ELP[V2Y-45],280 nm = 0.799 L/(g·cm) [ 32 ], and ε Casein,280 nm = 0.73 L/(g*cm) [ 22 ].…”