Design of core streptavidin SbsB fusion constructs
We used a similar cloning strategy as described by Moll et al. in 2002 to create this construct. The sequence of SbsB without the first SLH domain (first 30 amino acids) was amplified using primers with an EcoRI and XhoI restriction site on the 5’ and 3’ end respectively. A synthetic Streptavidin construct, codon optimized for E. coli expression, was created which lacks the first 16 amino acids. Therefore, only contains the sequence needed for biotin binding. The sequence contained a 5’ NdeI and a 3’ EcoRI restriction site.
As backbone we used the E. coli expression vector pET28a(+). An intermediate vector of pET28a(+) and Streptavidin was created via NdeI and EcoRI restriction. This vector was transformed into E. coli DH5α. Isolated Intermediate Vector was then ligated with our SbsB construct through EcoRI and XhoI digestions. This way of cloning was used to attach an N-terminal His-tag to our construct already encoded on the backbone.
Figure 2. pET28a(+):Strav+SbsB vector map.
Design of PS2 fusion constructs
In our project we want to create fusion proteins of CFP/YFP and the S-Layer protein PS2 from Corynebacterium glutamicum (Fig 1). These constructs can be used for FRET measurements to proof proximity induced through S-Layer self-assembly.
We wanted to create fusion proteins capable of attachment to the cell wall of C. glutamicum. Therefore, neither N- nor C- terminal fusions were possible due to the distinct properties of those segments. The tertiary structure of PS2 is not known, which makes the design difficult. We decided to use flexible G4S linkers in front and after the fluorescence proteins and created multiple constructs of CFP/YFP after 1500, 300 and 600 amino acids.
N-terminal fusion proteins are a possibility if the protein will be expressed in E. coli. This assumption is made due to other already described S-Layer fusion proteins (e.g. Core Streptavidin and SbsB) .
Figure 1. Different approaches of PS2 fusion proteins.
Green color stands for PS2 sequences, yellow or blue are fluorescence proteins and grey describe linker sequences.