A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.
A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.
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DOI：
10.1002/bit.25971
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发表时间：
2016-09
影响因子：
3.8
通讯作者：
Ward J
中科院分区：
文献类型：
--
作者：
Hassan S;Keshavarz-Moore E;Ward J
随着质粒DNA载体在基因治疗中的前景的最近复兴，一种新的合成生物学方法被用于提高质粒DNA的数量、产量和质量。质量通过超螺旋百分比和超螺旋密度以及提高发酵中的分离稳定性来衡量。我们检验了添加强促旋酶结合位点（SGS）会增加DNA促旋酶介导的质粒超螺旋的假设。将来自三种不同复制子（Mu噬菌体和两种质粒pSC 101和pBR 322）的SGS插入质粒pUC 57中。将这些不同大小的变异体转化到E. coli DH 5 α，测定其超螺旋特性和分离稳定性。在pUC 57-SGS中发现超螺旋密度增加了36%，但仅当SGS来源于Mu噬菌体并且是该片段的较大尺寸版本时。这些结果也在发酵规模下得到证实。超螺旋单体的总百分比保持在85- 90%。与pUC 57相比，pUC 57-SGS的质粒产量也增加了两倍。pUC 57-SGS显示出比pUC 57-cer和pUC 57更高的分离稳定性，证明SGS位点的进一步潜在优势。这些发现将增加质粒DNA载体在质粒DNA生产中的潜力。Biotechnol. Bioeng. 2016;113：2064-2071。© 2016作者。生物技术和生物工程出版的威利期刊，公司。
With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.