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 32P-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.

中文翻译：

---

从表达人干扰素-γ的大肠杆菌细胞获得的包涵体中的核酸。

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。尽管已经证明IBs包含功能性蛋白质和蛋白质聚集体，但是它们的异质性和细菌来源的有害污染物阻碍了它们作为药物的直接应用。因此，与可溶性物种的生产一起，IBs仍然是用于医疗应用的重组蛋白生产的主要来源。IB的质量和组成影响重组蛋白的重折叠产量和进一步纯化。核酸是IBs的真正成分还是伴随的杂质的知识，是了解IBs形成和开发重组蛋白重新折叠和纯化的最佳方法的前提。从过量表达人干扰素-γ（hIFNγ）（具有治疗用途的蛋白质）的大肠杆菌中分离出的IB被用作模型。在标准条件下从用hIFNγ表达质粒转化的大肠杆菌LE392细胞中分离出IB，并通过在蔗糖垫上离心进一步纯化，然后进行超声处理，并用非变性浓度的尿素洗涤。通过SDS-PAGE凝胶电泳和平行微生物学测试来评估纯化效率，以检测残留完整细菌的存在。苯酚/氯仿萃取表明，高度纯化的IBs同时含有DNA和RNA。通过紫外光谱和琼脂糖凝胶电泳结合酶处理和杂交研究了后者。观察到DNA作为主要在250至1000bp范围内的扩散部分。TRIzol®分离的RNA也显示出明显的分子异质性。与32P标记的寡核苷酸杂交显示，这些IB包含rRNA，并且富含hIFNγmRNA。这项研究提出的结果表明，核酸可能是IB中的固有成分，而不是共沉淀的杂质。我们假设核酸是重组蛋白聚集和IBs形成的积极参与者，这些IBs起源于微生物细胞工厂的转录和翻译机制。为了确定这一概念，还需要进一步研究。