Escherichia coli ( E. coli ) is often used as a host to produce a variety of recombinant proteins. However, the direct production of soluble and active protein containing disulfide bonds in E. coli remains a major challenge. In this study, we established a new, innovative soluble protein expression system based on the synergistic action of elastin-like polypeptide (ELP) and intein. Kinds of proteins including nanobodies, hIL-3 (Human Interleukin-3) analogue and AVN (the N terminal of A. ventricosus MiSp) with amino acid sequences that are normally express as insoluble form or difficult to express could be successfully expressed in soluble form in E. coli with this system. The soluble expression of the target proteins in the cytoplasm was achieved by the fusion of the solubility enhancer ELP, and the fusion of intein allowed the target proteins to be directly separated from the solubility enhancers in a mild enzyme-free environment. Through a one or two-step operation, nanobodies with high affinity activity, IL-3 analogue with bioactivity and soluble AVN were easily purified. This simple and efficient soluble expression system could thus be widely applied to facilitate the expression of difficult-to-express proteins.
Recombinant adeno-associated viral vectors (rAAVs) can effectively deliver transgene to the nervous system. The selection of AAV serotype and promoter significantly influences the dynamics of the transgene expression, including its strength and cell-specificity. Previous studies demonstrated that in neonatal mice, the intramuscular (IM) injection of the rAAV2-retro vector could efficiently deliver transgene to lower motor neurons (LMNs) of the brainstem and spinal cord. However, the best promoter for the expression of transgene in the central neural system (CNS) using rAAV2-retro remains undetermined. This study compared five commonly used promoters, including mouse phosphoglycerate kinase (mPGK), CMV early enhancer/chicken β-actin/short β-globulin intron (CAG), human cytomegalovirus (hCMV), chicken β-actin (CBA), and human synapsin (hSyn) promoters. The IM (unilateral gastrocnemius muscle) injection of rAAV2-retro vectors packaged with the reporter constructs containing each promoter was performed in the newborn C57BL/6J mice. The levels of gene expression and the types of cells were examined using the light-sheet illumination imaging technique and confocal microscopy. Our findings revealed that rAAV2-retro primarily targeted the brainstem and spinal cord within the CNS. Among the five promoters tested, CAG and hCMV showed the highest gene expression. Almost all the transduced cells were identified as LMNs. Additionally, gene expression driven by hCMV was found to be dependent of the inclusion of WPRE and β-globin intron elements. Importantly, none of the promoters induced hepatotoxicity, ensuring the safety of rAAV2-retro-mediated expression. This study provided valuable insights for optimizing the rAAV2-retro-mediated gene delivery system to LMNs in the brainstem and spinal cord, which might have potential implications for research on motor neuron-related diseases.
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