Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.

由于Sec和Tat介导的细菌脂质修饰在细胞功能，膜靶向和ELISA，生物传感器，无佐剂疫苗，脂质体药物递送等生物技术应用中起着至关重要的作用，因此它们是重要的翻译后过程。分泌和脂质修饰机械与相关过程的紧密结合将有助于阐明这一重要的生物学事件，并将其用于工程应用。此外，需要对工程化脂蛋白的膜靶向，增溶和纯化的简便性进行系统且令人信服的研究，以帮助科学家容易地应用这种新的蛋白质工程工具。因此，在这项研究中，我们系统地研究了重组表达，易位，增溶和纯化三种白斑综合症病毒（WSSV）蛋白ICP11，VP28和VP281。我们的研究表明，脂质修饰和分泌过程紧密相关，以至于折叠动力学和目标蛋白信号序列之间的不匹配可能导致转录-翻译解偶联或翻译中止。通过Tat途径表达为脂蛋白的蛋白质靶向内膜，可实现相当大的富集。然后使用一步固定化金属亲和色谱将这些带有His标签的蛋白纯化为无洗涤剂形式的表观同质性。这项研究在脂蛋白生物发生方面有有趣的发现，从而扩大了这种独特的翻译后蛋白质工程工具的范围，可用于获得无洗涤剂的纯净，