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Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP 5 to ELP 50 was constructed using recursive directional ligation by plasmid reconstruction. ELP 50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP 50 , which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP 50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP 50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP 50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP 50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP 50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The ELP fusion proteins were successfully recovered from total soluble protein with adding 2.75 M NaCl and three rounds of inverse transition cycling (ITC). The hIFN-γ was also separated from ELP with Enterokinase cleavage of the fusion protein and recovered by ITC. Inverse transition analysis indicated that the hIFN-γ-ELP variants aggregate above their inverse transition temperature and at high ionic strength. Investigation of glycosylation revealed that fused or unfused hIFN-γ proteins are N-glycosylated in different cellular locations. Moreover, N-glycosylation analysis and bioassay showed that fusion to ELP does not disturb glycosylation process and antiviral activity of hIFN-γ.

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.