Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction

Abstract

Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

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

“…During the hot spin, nucleic acid contamination could be further reduced compared to the HSP step and the highest purity (88.0% ± 4.4%) of all methods applied was achieved. Nevertheless, nucleic acids could not be reduced as efficiently for IMAC and may require additional processing for ITC-purified samples ( Meyer and Chilkoti, 1999 ; Lim et al, 2007 ; Verheul et al, 2018 ). Still, highly efficient ITC purification is limited to ELPs without aggregation effects at low temperatures in relevant concentration ranges and buffer systems.…”

“…To date, studies comparing the performance of purification processes of biomacromolecules TAGGED WITH either an ELP or polyhistidine showed a comparable product yield ( Meyer et al, 2001 ; Trabbic-Carlson et al, 2004 ; Hu et al, 2010 ). Additionally, new purification processes such as microfiltration of ELP fusion proteins ( Ge et al, 2006 ) and organic extractions ( Verheul et al, 2018 ; Mills et al, 2019 ) were introduced. Still, IMAC and ITC are common downstream processes for ELPs as purification target, which are possibly suitable for large-scale productions.…”

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium

“…Cells were fermented in terrific broth media supplemented with 1 mM NaCl for 24 h at 37 °C without IPTG induction. After centrifugation, 1 g of biomass (cell pellets) was resuspended in a 4 mL of 1:1 mixture of 1-butanol and ethanol (i.e., mixture of 8 mL 1-butanol and 8 mL ethanol was directly added to 4 g of cell pellet) [ 42 ]. Cell pellets were resuspended thoroughly by vortexing (10 s) and left under constant agitation at room temperature for 15 min.…”

Broadening Approaches in Nanoparticle Synthesis Through Elastin-Like Polypeptides

“…can be solubilized in organic solvents with different polarities. (Verheul et al, 2018;Zhao et al, 2020) This property has been exploited by researchers not only to perform ELPs purification but also for the fabrication of biomedical devices through processes as electrospinning or 3D bioprinting. (Putzu et al, 2019;Dai et al, 2021) Specially revealing is the work carried out by Elsharkawy and colleges where ELPs are used to control the biomineralization of apatite nanocrystals by tuning the disorder-order ratio.…”