The SCP-2L gene was codon-optimised for E. coli and prepared by gene synthesis. Amber stop codons were introduced at A100, V83 or Q111 by site-directed mutagenesis. E. coli BL21 (DE3) cells were co-transformed with the pEVOL-BpyAla plasmid, which contains the orthogonal MjTyrRS/MjtRNA Tyr genes , and the pET28 plasmid carrying the SCP gene with a C-terminal TEV cleavable His6 tag. The genes were expressed in the presence of 0.5 mM BpyAla and the resulting proteins purified by Ni-affinity chromatography with the His6 tag removed using TEVprotease. Typical yields were 5-15 mg L -1 in LB media, representing a decrease of 20-80% compared to the yields of SCP-2L without BpyAla. Proteins containing unnatural amino acids are known to have lower expression yields, due to incomplete stop codon supression giving truncated protein. Mass spectrometry on the purified proteins confirmed the successful incorporation of BpyAla at residue positions 100, 111, or 83 (Figure and ESIa Fig. ). The mass of the BpyAla proteins is not 46 Da lower than the SCP CBpy proteins as might be expected due to the absence of the -CH2S-linker, but higher due to the presence of additional amino acids from the TEV site remaining at the C-terminus.).