High-Yield Recombinant Expression and Purification of Marginally Soluble, Short Elastin-Like Polypeptides

Abstract

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.

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

“…Extraction with nBuOH:Ace (Figure S7) was also effective with the 15 min incubation. Although reports have indicated exceptionally high yields for ELP production under IPTG induction, 31 the yield in our case was significantly lower for an equivalent cell mass. The reduced yield may be due to ELP entrainment within inclusion bodies.…”

“…Then, the cell suspension was probe tip sonicated, on ice, with a 1/8″ microtip and a 30 % duty cycle for 15 min. One of two lysis buffers were used for chemical lysis: homemade buffered urea 31 or commercial B-PER (Thermo Fisher Scientific). For the urea method, cells were thoroughly resuspended in lysis buffer (8 M urea, 100 mM NaH 2 PO 4 , 10 mM Tris, pH 8.0; 4 mL per 1 g wet pellet weight) and incubated at 20 °C for 1 h on a shaker.…”

“…For example, non-protein macromolecular contaminants, such as nucleic acids, can be problematic for the ITC process and are commonly co-purified with the target ELP. 27,28,31 Although a nucleic acid precipitation step with branched polyethylenimine (bPEI) is often effective before ITC, it increases the processing cost and time, as well as being incompatible with certain ELP constructs. 31,32…”

Self-assembly/disassembly hysteresis of nanoparticles composed of marginally soluble, short elastin-like polypeptides

“…ELPs were synthesized according to Bahniuk et al [ 7 ]. Briefly, the initial ELP genes were purchased through Integrated DNA Technologies (Coralville, IA, USA) and were concatemerized together in a pUC-19 cloning vector (Bio Basic, Ontario, Canada) using a modified recursive directional ligation procedure.…”

“…This study begins to address these unknown parameters while simultaneously expanding upon the existing body of knowledge regarding systematically-studied behaviour of ELPs. The effects of chain length, amino acid chemistry and protein concentration were re-examined using a novel suite of short, highly hydrophobic ELPs developed by our group [ 7 ]. ELPs composed of 20, 40, 80 or 160 pentapeptide repeats with leucine in the guest amino acid position, as well as a valine-containing 40-mer control, were studied across a range of concentrations and temperatures using dynamic light scattering (DLS), zeta-potential and transmission electron microscopy (TEM).…”