In vivo self-aggregation and efficient preparation of recombinant lichenase based on ferritin / 生物工程学报

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.

Factors and mechanism influencing elastin-like polypeptides self-assembled into micron-sized particles / 生物工程学报

Many factors influence the elastin-like polypeptides (ELPs) self-assembled into micron-sized particles. However, few efforts were made to investigate these factors. Using the ELPs [KV8F]n as the target, we studied systematically the factors with the dynamic light scattering. Our results show that the particle size increased and the uniform of particles decreased with the increase of the molecular weight. The analysis of size variation in self-assembled ELPs in response to changes in salt concentration indicated that the size increased with increasing the salt concentration, and the opposite response was observed when the concentration was above 0.4 mol/L. Under these conditions, the particles are micron-sized and larger than 1.1 μm. However, when the fusions containing the same ELPs and xylanase or 1,3-propanediol oxidoreductase, the size of the self-assembled ELPs particles decreased dramatically, which was only about 1/10 of that of the free ELPs. We proposed that the solvent accessible charged area of the enzymes could interact with the ELPs, the sterical hindrance of the enzymes prevent the aggregation of the ELPs. This might be the most important parameter in altering the particle size sharply.

De novo design, non-chromatographic purification and salt-effect of elastin-like polypeptides / 生物工程学报

Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.