Bacterial lipid modification of proteins requires appropriate secretory signals even for expression – Implications for biogenesis and protein engineering. (September 2014)

• Record Type: Journal Article
• Authors: Kumar, Subramani; Balamurali, M. M.; Sankaran, Krishnan
• Is Part Of: Molecular membrane biology. Volume 31:Number 6(2014)
• Page Start: 183
• Page End: 194
• Publication Date: 2014-09
• Subjects: Membranes (Biology) -- Periodicals; Biochemistry -- Periodicals; Cell membranes -- Periodicals; Molecular biology -- Periodicals; 571.64
• DOI: 10.3109/09687688.2014.943819
• Languages: English
• ISSNs: 0968-7688

Abstract

<abstract> <title>Abstract</title> <p>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.</p> </abstract>