Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

A general controllable release amplification strategy of liposomes for single-particle collision electrochemical biosensing.

As an important component of the COVID-19 mRNA vaccines, liposomes play a key role in the efficient protection and delivery of mRNA to cells. Herein, due to the controllable release amplification strategy of liposomes, a reliable and robust single-particle collision electrochemical (SPCE) biosensor was constructed for H9N2 avian influenza virus (H9N2 AIV) detection by combining liposome encapsulation-release strategy with immunomagnetic separation. The liposomes modified with biotin and loaded with platinum nanoparticles (Pt NPs) were used as signal probes for the first time. Biotin facilitated the coupling of biomolecules (DNA or antibodies) through the specific reaction of biotin-streptavidin. Each liposome can encapsulate multiple Pt NPs, which were ruptured under the presence of 1 × PBST (phosphate buffer saline with 0.05% Tween-20) within 2 min, and the encapsulated Pt NPs were released for SPCE experiment. The combination of immunomagnetic separation not only improved the anti-interference capabilities but also avoided the agglomeration of Pt NPs, enabling the SPCE biosensor to realize ultrasensitive detection of 18.1 fg/mL H9N2 AIV. Furthermore, the reliable SPCE biosensor was successfully applied in specific detection of H9N2 AIV in complex samples (chicken serum, chicken liver and chicken lung), which promoted the universality of SPCE biosensor and its application prospect in early diagnosis of diseases.