Part:BBa_K5487002

Design Notes Function: The Lpp-OmpA fusion protein serves as a versatile tool for the surface display of various peptides and proteins on the outer membrane of bacterial cells, such as Escherichia coli. The Lpp signal peptide ensures the correct localization and secretion of the fused protein to the bacterial outer membrane, while OmpA provides a stable anchor and a high density of presentation.

Mechanism:

Signal Sequence Recognition: The Lpp signal peptide is recognized by the cell's secretion machinery, directing the fusion protein to the secretion pathway. Translocation: The protein is translocated across the cell membrane, with the signal peptide being cleaved off in the process. Outer Membrane Anchoring: The OmpA domain anchors the protein to the outer membrane, ensuring its stable display on the cell surface. Optimal Conditions:

Temperature: The optimal growth temperature for bacterial cells displaying the Lpp-OmpA fusion protein is typically around 37°C. Media: Bacteria should be cultured in appropriate media that support robust growth and protein expression, such as Luria-Bertani (LB) broth. Application:

Protein Display: Used for the display of heterologous proteins on bacterial surfaces for various applications, including vaccine development, biocatalysis, and biosensing. Cell Surface Engineering: Employed in the engineering of bacterial cell surfaces for biotechnological applications, such as bioremediation and bioproduction. Immunological Studies: Utilized for the presentation of antigenic determinants to study immune responses and develop diagnostic tools. Usage in Projects: To use the Lpp-OmpA fusion protein in a project, follow these steps:

Cloning: Clone the gene of interest (GOI) between the Lpp and OmpA coding sequences, ensuring in-frame fusion. Transformation: Transform the construct into a suitable bacterial host. Expression: Induce expression under appropriate conditions to display the fusion protein on the bacterial surface. Verification: Confirm surface display using techniques such as immunofluorescence, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). Safety Considerations:

Handle bacterial cultures and recombinant DNA with appropriate biosafety precautions. Use personal protective equipment (PPE) and follow good microbiological practices. Storage and Stability:

Store bacterial glycerol stocks at -80°C for long-term preservation. Maintain cell cultures under stable conditions to prevent loss of surface-displayed protein.

Source Component Origin:

Lpp (Lipoprotein Signal Peptide): The Lpp signal peptide is derived from the N-terminal sequence of a lipoprotein found in various bacterial species, often Escherichia coli. It is used for the secretion and localization of proteins to the bacterial cell membrane.

OmpA (Outer Membrane Protein A): OmpA is a well-characterized outer membrane protein from E. coli. It serves as a stable anchor for the fusion partner on the outer membrane of Gram-negative bacteria.

Genomic Sequence:

The Lpp-OmpA construct is typically synthesized or codon-optimized for expression in the host organism and may not directly correspond to a specific wild-type gene sequence from the bacterial genome. Instead, it is a composite part created through genetic engineering techniques. Engineering Process:

Design: The Lpp-OmpA fusion is designed by genetic engineers who select the signal peptide and anchoring protein sequences based on their known secretion and surface localization properties. Synthesis: The DNA sequence encoding the Lpp-OmpA fusion is chemically synthesized to ensure accuracy and optimize expression in the chosen host. Cloning: The synthesized sequence is cloned into an expression vector that allows for the insertion of a gene of interest (GOI) between the Lpp and OmpA sequences, creating an in-frame fusion. Expression System:

The Lpp-OmpA part is commonly expressed in bacterial systems such as E. coli due to their ease of manipulation and high expression capabilities. The choice of host can vary depending on the project's requirements. Purification and Verification:

After expression, the fusion protein is typically anchored to the outer membrane of the bacterial cells, and its presence is verified through various biological assays. Application in Synthetic Biology:

In synthetic biology, the Lpp-OmpA part is a standard component used for the surface display of proteins, facilitating applications such as whole-cell biocatalysis, biosensing, and vaccine development.

References Vahed M, Ramezani F, Tafakori V, Mirbagheri VS, Najafi A, Ahmadian G. Molecular dynamics simulation and experimental study of the surface-display of SPA protein via Lpp-OmpA system for screening of IgG. AMB Express. 2020 Sep 3;10(1):161. doi: 10.1186/s13568-020-01097-1. PMID: 32880759; PMCID: PMC7471224.

Richins RD, Kaneva I, Mulchandani A, Chen W. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase. Nat Biotechnol. 1997 Oct;15(10):984-7. doi: 10.1038/nbt1097-984. PMID: 9335050.

Peschke T, Rabe KS, Niemeyer CM. Orthogonal Surface Tags for Whole-Cell Biocatalysis. Angew Chem Int Ed Engl. 2017 Feb 13;56(8):2183-2186. doi: 10.1002/anie.201609590. Epub 2017 Jan 20. PMID: 28105787.