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

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.


S2. Quantitative analytical methods tested
Cation exchange chromatography Analytical cation exchange chromatography was conducted on a Vanquish Flex -R2L UHPLC System (Thermo Fisher Scientific) equipped with a TSKgel Q-STAT column (Tosoh Bioscience, Tokyo, JP). The column was equilibrated for 2 min with low salt buffer (10 mM citrate containing 4 M urea, pH 3) before a 10 µl sample injection. A column wash for one more minute with low salt buffer was performed before a 6 min linear gradient from 0 to 100 % with high salt buffer (10 mM citrate containing 4 M urea and 0.75 M sodium chloride, pH 3), which was hold for 4 minutes. Finally, the column was re-equilibrated with a 2 min wash using low salt buffer. All samples were prior filtered through 0.45 µm cellulose acetate filter (Pall Corporation, New York, US-NY). An initially via two cycles of ITC purified sample was applied to determine the ELP[V2Y-45] retention time, before process solution of the IMAC feed was loaded onto the column. In order to evaluate the purity of the ELP[V2Y-45] elution peak, it was fractionated and prepared for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF-MS). Therefore, Vivaspin® ultrafiltration units (Sartorius Stedim Biotech, Göttingen, DE) with a molecular weight cut off of 10 kDa were used to reach a final concentration of at least 2 mg/ml total protein concentration. Meanwhile, a buffer exchange towards 10 mM ammonium bicarbonate was performed by three centrifugation cycles. Molecular weight analysis was performed as described in the main text. Figure S2 -Intensities of the mass to charge ratio (m/z) of the fractionated, concentrated and rebuffered elution of the UHPLC elution peak containing ELP [V2Y-45]. Four peaks were identified by the spectrometer, three in a range below 16000 m/z and a main peak at 21594.203 m/z corresponding to ELP [V2Y-45].
Besides ELP [V2Y-45], three more proteins with a size below 16 kDa could be traced in the elution peak (see Figure S2). Therefore, no quantitative analysis of the target molecule could be reached by this analytical method.

Size Exclusion chromatography
Size exclusion chromatography can be conducted using urea containing buffer systems. Here, initially via two cycles of ITC purified ELP [V2Y-45] as well as two model proteins BSA (Merck KGaA) and lysozyme from chicken egg white (Hampton Research, Aliso Viejo, US-CA) were used as references for host-cell protein (HCP) contamination. All proteins were separately prepared with a concentration of 2 mg/ml in a 20 mM Tris (Tris(hydroxymethyl)aminomethane, Merck KGaA) buffer pH 8 containing 4 M urea, which was also used as running buffer in the SEC method. A constant flow rate of 0.3 ml/min was applied to a Zenix SEC-300 column (Sepax Technologies, Newark, US-DE) which was implemented in a Vanquish Flex -R2L UHPLC system. Thereby, two peaks could be observed for ELP [V2Y-45]. The main peak after a retention time of 4.9 minutes covered 97.9 % of total area with a peak shoulder. A smaller peak (2.1 % of the total peak area) could be traced after 2.9 minutes. As one model protein, BSA showed multiple peak behavior and its signal overlapped with the ELP main peak. The second model protein lysozyme showed a pronounced peak tailing. Due to the unsharp ELP peak and difficult model protein behavior, this approach was not further assessed with process solution.

Reversed-phase chromatography (RP)
In preliminary experiments, ELP[V2Y-45] was dissolved with a concentration of 1 mg/ml in common RP running buffers. Thereby, it showed to be insoluble in isopropanol, hexane and acetonitrile without further addition of urea. Since RP separation is based on hydrophobicity and the presence of urea affects hydrophobic interactions no further tests were conducted.

Capillary electrophoresis
Capillary electrophoresis was conducted on a Caliper LabChip®GX II device (PerkinElmer, Waltham, US-MA) according to manufacturer`s protocol. Since ELP[V2Y-45] precipitated during sample preparation and no results could be obtained, sample preparation was adapted by dissolution of the analyzed sample in 20 mM Tris buffer pH 8 containing 4 M urea. As verification that this adaption does not influence analytical quality, lysozyme was used as a protein standard, which showed similar results for both sample preparation methods. However, for ELP[V2Y-45] which was previously purified via two cycles of ITC, estimated protein size was above 52 kDa and no defined single peak could be identified. For an HCP containing sample, HCP signal overlayed the fluorescence signal of the ELP[V2Y-45]. Therefore, this method was not applicable for quantitative or qualitative analysis.

S4. Preparation of starting material
Several proteins in the 3.5 to 97 kDa size range are present in the cell lysate. Beside other protein bands, a clearly pronounced band at approximately 26 kDa is observable in the lysate centrifugation pellet, while this band does not stand out in the lysate centrifugation supernatant. The addition of a buffer containing 4 M Urea to the resulting pellet solubilized the inclusion bodies containing the target molecule. After a following centrifugation, the band at 26 kDa in the supernatant is more clearly pronounced than in the centrifugation pellet, while other HCP bands are more pronounced in the pellet compared to the supernatant.

Figure S4 -SDS-PAGE analysis of the start material generation. An Invitrogen TM Mark12 TM Unstained Standard (lane S) was used and the target molecule is indicated by arrows. Molecular weights of selected proteins contained in the standard are shown on the left. The lanes are: cell lysate (lane 1); supernatant (lane 2) and pellet (lane 3) after the lysate centrifugation; the generated start material (lane 4) and centrifugation pellet (lane 5) after the inclusion body dissolving.
Table S1 -A260/A280 ratios during the preparation of the start material for all following process steps. The lysate in an aqueous buffer without urea was measured before and after centrifugation. The lysate pellet was resuspended in a buffer system containing 4 M urea for inclusion body dissolution and got centrifuged again (n = 3).