Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.
Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.
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DOI：
10.1186/s12934-021-01719-8
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发表时间：
2021-12-20
影响因子：
6.4
通讯作者：
Li S
中科院分区：
文献类型：
--
作者：
Wang K;Zhou L;Chen T;Li Q;Li J;Liu L;Li Y;Sun J;Li T;Wang Y;Kong Z;Zheng Q;Zhang J;Yu H;Gu Y;Xia N;Li S
与大肠杆菌的生长特性相关的各种优点证明了它们在基因工程疫苗生产中的使用是合理的。然而，内毒素污染、质粒载体不稳定性和对抗生素补充的要求是成功生产对于工业规模应用安全的重组蛋白的常见瓶颈。为了克服这些缺点，我们专注于中断参与脂多糖（LPS）合成的几个关键基因的表达，脂多糖是一种经常导致重组蛋白毒性的内毒素，以消除内毒素污染并用E.杆菌在8个与LPS合成相关的潜在靶基因中，我们成功构建了7个LPS生物合成缺陷型重组菌株，以减少LPS的产生。这些修饰的E.大肠杆菌菌株的产量比野生型菌株低约两个数量级。进一步研究发现，6个位点lpxM、lpxP、lpxL、eptA、gutQ和kdsD-1适合于HPV L1蛋白的染色体整合表达。我们发现，与基于质粒的表达的更可变的蛋白质生产相比，表达盒的单拷贝在长期无菌培养期间赋予稳定的表达。在大规模发酵中，我们发现与亲本ER 2566菌株相比，携带3至5个拷贝的表达盒的重组菌株具有1.5至2倍高的总体表达沿着较低的内毒素水平。最后，我们设计并构建了9个重组E.大肠杆菌菌株用于随后生产具有所需纯度、VLP形态和抗原性的HPV 9价衣壳蛋白。重组E. coli ER 2566菌株通过表达盒的染色体整合具有生产9价HPV候选疫苗的潜在用途，其具有显著降低的残留内毒素水平。我们的研究结果为重组大肠杆菌提供了一种新的策略。大肠杆菌菌株的构建、工程化以及合适的重组蛋白药物的开发。在线版本包含补充材料，可通过10.1186/s12934-021-01719-8获得。
The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs. The online version contains supplementary material available at 10.1186/s12934-021-01719-8.
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影响因子：
5
作者：
Kito, M;Itami, S;Shibui, T
通讯作者：
Shibui, T
DOI：
10.1007/978-1-4939-6887-9_1
发表时间：
2017-01-01
期刊：
HETEROLOGOUS GENE EXPRESSION IN E. COLI: METHODS AND PROTOCOLS
影响因子：
--
作者：
Gileadi, Opher
通讯作者：
Gileadi, Opher
影响因子：
14
作者：
Gorbet, MB;Sefton, MV
通讯作者：
Sefton, MV
影响因子：
3.2
作者：
Sezonov, Guennadi;Joseleau-Petit, Daniele;D'Ari, Richard
通讯作者：
D'Ari, Richard
DOI：
10.1196/annals.1409.002
发表时间：
2007-01-01
期刊：
FRANCISELLA TULARENSIS: BIOLOGY, PATHOGENICITY, EPIDEMIOLOGY, AND BIODEFENSE
影响因子：
--
作者：
Titball, Richard W.;Sjostedt, Anders;Nano, Francis E.
通讯作者：
Nano, Francis E.