Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, Academic Georgi Bonchev Str., Blok 21, 1113, Sofia, Bulgaria.
Microb Cell Fact. 2020 Jul 11;19(1):139. doi: 10.1186/s12934-020-01400-6.
Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model.
IBs were isolated from E. coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol also demonstrated a substantial molecular heterogeneity. Hybridization with P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.
The results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.
包涵体（IBs）是重组细菌细胞中的蛋白质聚集体，主要包含目标重组蛋白。尽管已经表明 IBs 包含功能性蛋白质以及蛋白质聚集体，但由于其异质性和源自细菌的有害污染物，它们直接作为药物的应用受到阻碍。因此，除了生产可溶性物种外，IBs 仍然是制造具有医疗应用的重组蛋白的主要来源。IBs 的质量和组成会影响重组蛋白的复性产率和进一步纯化。核酸是否是 IBs 的真正组成部分还是伴随的杂质，是理解 IBs 形成的前提，也是开发用于重组蛋白复性和纯化的优化方案的前提。本文以表达治疗性蛋白人干扰素-γ（hIFNγ）的大肠杆菌 LE392 细胞为模型，研究了 IBs 的组成。
在标准条件下，从表达 hIFNγ 的质粒转化的大肠杆菌 LE392 细胞中分离出 IBs，然后通过在蔗糖垫上离心、多次超声处理和用非变性浓度的脲洗涤进一步纯化。通过 SDS-PAGE 凝胶电泳和并行微生物学测试评估纯化效率，以检测残留的完整细菌。酚/氯仿抽提表明高度纯化的 IBs 既含有 DNA 又含有 RNA。后者通过 UV 光谱学和琼脂糖凝胶电泳与酶处理和杂交相结合进行研究。DNA 主要在 250 到 1000 bp 范围内呈现弥散部分。用 TRIzol 提取的 RNA 也表现出显著的分子异质性。用 P 标记的寡核苷酸杂交表明 IBs 含有 rRNA，并富含 hIFNγ mRNA。
本研究的结果表明，核酸可能是 IBs 的固有组成部分，而不是共沉淀的杂质。我们假设核酸是重组蛋白聚集和 IBs 形成的活性参与者，这些 IBs 起源于微生物细胞工厂的转录和翻译机制。需要进一步的研究来证实这一观点。
