title: Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.
abstract: 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.
authors.affiliation: Centre for Biotechnology, Anna University , Chennai , India.
authors.forename: Subramani
authors.initials: S
authors.lastname: Kumar
authors.forename: M M
authors.initials: MM
authors.lastname: Balamurali
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sankaran
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015696:Gene Products, tat; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D033561:Penaeidae / Q000821:virology*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D046848:White spot syndrome virus 1 / Q000235:genetics* / Q000378:metabolism
chemical_list: D015696:Gene Products, tat; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014764:Viral Proteins; D019308:Palmitic Acid
keywords: Lipoprotein biogenesis; Sec; Tat; WSSV proteins; membrane targeting; post-translation; protein engineering; purification; secretion; solubilization; uncoupling