Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 66-454, Cambridge, MA 02139, USA.
Microb Cell Fact. 2012 Aug 13;11:107. doi: 10.1186/1475-2859-11-107.
There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production.
In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C.
Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.
近年来，由于批准了几种兽医 DNA 疫苗、正在进行的基于人 pDNA 的治疗方法的临床试验以及在佐剂和传递载体方面的重大进展，这些进展有助于克服早期疗效不足，基于质粒 DNA（pDNA）的生物制药重新引起了人们的兴趣。随着人们对这种兴趣的增加，人们对高产量、具有成本效益的制造工艺的需求也在增加。为此，载体工程是一种提高质粒生产的有前途的策略。
在这项工作中，我们构建了一种新的 DNA 疫苗载体 pDMB02-GFP，其中包含了失控 R1 复制起点。失控复制表型应该会导致质粒拷贝数在 30°C 到 42°C 的温度转换后扩增。然而，使用大肠杆菌 DH5α 作为宿主，我们观察到在 30°C 恒温和培养时，pDMB02-GFP 的产量最高，最大产量约为 19 mg pDNA/g DCW。通过测量 R1 复制起始蛋白 RepA 的 mRNA 和蛋白水平，我们确定 RepA 可能在 42°C 时限制了 pDMB02-GFP 的产量。通过将 repA 的起始密码子从非最佳 GTG 更改为 ATG，构建了一个突变质粒 pDMB-ATG。在 DH5α[pDMB-ATG]的培养物中，与观察到的 pDMB02-GFP 相比，温度诱导的质粒扩增更为剧烈，并且 RepA 蛋白在 42°C 下比 pDMB02-GFP 的培养物中可检测到的时间长几个小时。
总的来说，我们已经证明了基于 R1 的质粒可以在不需要温度转换的情况下产生高产高质量的 pDNA，并且为进一步研究这一类载体在质粒 DNA 生产方面奠定了基础。
