<p class="para">Now that our plasmid design has been verified using the sequencing results and the SPAAC reaction has been verified using UV-VIS and LCMS, it is time to check whether the plasmids result in the wanted protein expression. To verify the expression of Clickable Outer Membrane Protein X (COMPx) and Y (COMPy), fluorescent labelled dibenzocyclooctyne (DBCO) groups, DBCO-PEG4-5/6-TAMRA (Figure 1), have been reacted with the incorporated p-azido-L-phenylalanine (pAzF).</p>
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<p class="para">In short, both membrane proteins had a TAG codon introduced for the Non Natural Amino Acid and a HA-tag for detection methods. For INPNC the TAG codon and the HA-tag were introduced at the C terminus of the protein (figure 1). The CPX proteins already contained a peptide library part at the N-terminus so the TAG codon was introduced in that part. This has been done by using Site Directed Mutagenesis. The HA-tag was introduced at the C-terminus (figure 2). After modification the proteins were renamed to Clickable Outer Membrane Protein x (COMPx), originally CPX, and COMPy, originally INPNC.</p>
In short, both membrane proteins had a TAG codon introduced for the Non Natural Amino Acid and a HA-tag for detection methods. For INPNC the TAG codon and the HA-tag were introduced at the C terminus of the protein (figure 1). The CPX proteins already contained a peptide library part at the N-terminus so the TAG codon was introduced in that part. This has been done by using Site Directed Mutagenesis. The HA-tag was introduced at the C-terminus (figure 2). After modification the proteins were renamed to Clickable Outer Membrane Protein x (COMPx), originally CPX, and COMPy, originally INPNC.
Figure 1. Gene design COMPy (INPNC)
Bibliography
[1] Click Chemistry Tools, B. T. (n.d.). Products: TAMRA DBCO. Retrieved July 23, 2014, from https://www.clickchemistrytools.com/products/click_chemistry_toolbox/azide_cycloalkyne_click_chemistry/cyclooctynes/fluorescent_probes/tamra-dbco-prob/
[2] Macey, M. G. (2007). Cell Sorting by Flow Cytometry. In Flow Cytometry: Principles and Applications (pp. 257 - 276). Humana Press.
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