title: Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli.
abstract: Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.
authors: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, P.O. Box 71345-1583, Iran.
authors: Mohammad
authors: M
authors: Sadraeian
authors: Mohammad Bagher
authors: MB
authors: Ghoshoon
authors: Milad
authors: M
authors: Mohkam
authors: Zeinab
authors: Z
authors: Karimi
authors: Sara
authors: S
authors: Rasoul-Amini
authors: Younes
authors: Y
authors: Ghasemi
mesh_terms: D058947:Bacterial Secretion Systems; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022603:Shiga Toxins / Q000235:genetics / Q000378:metabolism*; D014765:Viral Vaccines / Q000235:genetics / Q000378:metabolism*
chemical_list: D058947:Bacterial Secretion Systems; D003470:Culture Media; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; D014765:Viral Vaccines; C059731:oncogene protein E7, Human papillomavirus type 16; C077239:stxB toxin