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• Michalik, Marcin; Djahanschiri, Bardya; Leo, Jack Christopher & Linke, Dirk (2022). An Update on “Reverse Vaccinology”: The Pathway from Genomes and Epitope Predictions to Tailored, Recombinant Vaccines. I Thomas, Sunil (Red.), Vaccine Design. Methods and Protocols, Volume 3: Resources for Vaccine Development. Second Edition. Humana Press. ISSN 9781071618912. s. 45–71. doi: 10.1007/978-1-0716-1892-9. Fulltekst i vitenarkiv Vis sammendrag

  In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the concept of reverse vaccinology. They all follow the same basic principles—mining available genome and proteome information for antigen candidates, and recombinantly expressing them for vaccine production. Some of the same principles have been used successfully for cancer therapy approaches. In this review, we focus on infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development.

• Meuskens, Ina; Michalik, Marcin; Chauhan, Nandini; Linke, Dirk & Leo, Jack Christopher (2017). A New Strain Collection for Improved Expression of Outer Membrane Proteins. Frontiers in Cellular and Infection Microbiology. 7. doi: 10.3389/fcimb.2017.00464. Fulltekst i vitenarkiv Vis sammendrag

  Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

• Michalik, Marcin; Rydmark, Marcella Orwick; Habeck, Michael; Alva, Vikram; Arnold, Thomas & Linke, Dirk (2017). An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins. PLOS ONE. 12(8). doi: 10.1371/journal.pone.0182016. Fulltekst i vitenarkiv Vis sammendrag

  An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved ‘folding core’ that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

• Michalik, Marcin; Djahanshiri, Bardya; Leo, Jack Christopher & Linke, Dirk (2016). Reverse Vaccinology: The Pathway from Genomes and Epitope Predictions to Tailored Recombinant Vaccines. I Thomas, Sunil (Red.), Vaccine Design Methods and Protocols: Volume 1: Vaccines for Human Diseases. Springer. ISSN 9781493933853. s. 87–106. doi: 10.1007/978-1-4939-3387-7_4. Fulltekst i vitenarkiv

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• Meuskens, Ina; Michalik, Marcin; Chauhan, Nandini; Linke, Dirk & Leo, Jack Christopher (2020). Corrigendum: A New Strain Collection for Improved Expression of Outer Membrane Proteins (Frontiers in Cellular and Infection Microbiology, (2017), 7, (464), 10.3389/fcimb.2017.00464). Frontiers in Cellular and Infection Microbiology. 10. doi: 10.3389/fcimb.2020.00220. Fulltekst i vitenarkiv
• Michalik, Marcin & Linke, Dirk (2015). Identification and evaluation of peptides for immunization against Pseudomonas aeruginosa. Fulltekst i vitenarkiv

Se alle arbeider i NVA