Braun H, Boller K, Löwer J, Bertling W M, Zimmer A
Institute for Pharmaceutical Technology, University of Frankfurt, Biozentrum, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
Biotechnol Appl Biochem. 1999 Feb;29 ( Pt 1):31-43.
The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength. The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4. These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA. The loading with DNA was pH-dependent. We observed the highest efficiency at pH 5. DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation.
此处描述的药物递送系统基于在大肠杆菌中表达的多瘤病毒衣壳蛋白VP1组装产生的类病毒体或衣壳样结构的特性。衣壳蛋白VP1作为融合蛋白表达，带有一个可完全去除的N端His6亲和标签。在低离子强度条件下进行亲和层析和因子Xa切割后，通过电子显微镜确认了重组VP1蛋白的五聚体形态。通过用(NH4)2SO4增加离子强度，可以从纯化的VP1五聚体诱导VP1类病毒体的自组装。这些VP1类病毒体颗粒在体外与反义寡核苷酸和质粒DNA进行了包封。DNA的包封依赖于pH值。我们观察到在pH 5时效率最高。用DNase I处理含有包封物质的颗粒表明，37-55%的结合寡核苷酸和1.5-1.8 kb双链DNA片段受到保护，不被降解。
