Bellini A V, Galli G, Fascetti E, Frascotti G, Branduzzi P, Lucchese G, Grandi G
Eniricerche S.p.A., Milan, Italy.
J Biotechnol. 1991 May;18(3):177-92. doi: 10.1016/0168-1656(91)90246-r.
The human IL-1 beta coding sequence derived from a cDNA library was inserted into two different plasmid expression vectors, pSM214 and pSM308, which promote the synthesis of recombinant products intracellularly and exocellularly, respectively. The hybrid constructs pSM261 and pSM320 were obtained. Bacillus subtilis SMS118 was transformed with these plasmids and the recombinant strains were grown in 1 litre bioreactors. Different growth conditions were analyzed to optimize yields both in terms of biomass and IL-1 beta production. In the pSM261-harbouring strain, IL-1 beta was synthesized in the cytoplasm to levels ranging from 1 to 2.7 mg g-1 of cells, corresponding to up to 40 mg l-1 of the culture. In contrast, SMS118(pSM320) was able to secrete 0.27 mg of natural IL-1 beta per g of cells (6.7 mg l-1 of culture). Processes for the purification of IL-1 beta from the supernatant and the biomass of the two cultures were also developed and compared in terms of yield and simplicity of the purification schemes. From our data it turns out that the route of intracellular expression is very efficient and superior to the one which results in secretion of IL-1 beta. This indicates that the use of B. subtilis as a recombinant host in biotechnology is not strictly dependent on its ability to secrete proteins into the culture medium.
从cDNA文库中获得的人白细胞介素-1β编码序列被插入到两种不同的质粒表达载体pSM214和pSM308中，这两种载体分别促进重组产物在细胞内和细胞外的合成。由此得到了杂交构建体pSM261和pSM320。用这些质粒转化枯草芽孢杆菌SMS118，并将重组菌株在1升生物反应器中培养。分析了不同的生长条件，以优化生物量和白细胞介素-1β产量方面的产量。在携带pSM261的菌株中，白细胞介素-1β在细胞质中合成，水平为每克细胞1至2.7毫克，相当于培养物中高达40毫克/升。相比之下，SMS118(pSM320)每克细胞能够分泌0.27毫克天然白细胞介素-1β(培养物中6.7毫克/升)。还开发了从两种培养物的上清液和生物量中纯化白细胞介素-1β的方法，并在产量和纯化方案的简便性方面进行了比较。从我们的数据来看，细胞内表达途径非常有效，优于导致白细胞介素-1β分泌的途径。这表明在生物技术中使用枯草芽孢杆菌作为重组宿主并不严格依赖于其将蛋白质分泌到培养基中的能力。
