Fig 1.
In-frame TAG codon replacement.
(A) Step 1 involves trinucleotide deletion followed by TAG donation using a combination of the engineered transposon MuDel [25] and the DNA cassette SubSeq [21] essentially as described previously [23] and outlined in B. The TAG substitution library is then cloned in front of the TEV-sfGFP cassette in plasmid pIFtag (S1 Fig). Step 2 outlines the selection for in-frame TAG substitutions. Initially, cells are grown in the absence of a nAA and non-fluorescent colonies selected; fluorescent colonies are removed at this stage as they are deemed not to have an in-frame TAG due to the generation of a full translation product. The second selection involves plating the selected colonies in the presence of nAA. Those cells that regain fluorescence suppress TAG termination due to nAA incorporation and thus produce sfGFP. (B) Alternate versions of the new SubSeq DNA cassette for donating TAG. The two alternatives are shown in the red boxes at the far left. (C) The two nAAs used in this study, p-azido-L-phenylalanine (azF) and p-iodo-L-phenylalanine (iodoF).
Fig 2.
Selection for in-frame TAG replacement.
(A) Schematic of the cytochrome (red) and sfGFP (green) fusion with the TEV cleavage site between the two shown. The residue targeted (K51) for replacement and the rational TAG replacements are shown. (B) cellular fluorescence of each variant in the absence (-nAA) and presence (+nAA) of either azF or iodoF. The wt refers to the cyt b562-sfGFP fusion without any TAG codon replacement. (C) SDS-PAGE (top panel) and Western blot (bottom panel) analysis of fusion protein expression in the soluble fraction of the cell lysate. The—v lane represents cells producing CG-wt in the absence of the pAB vector. The + and—lanes refer to the presence or absence of the nAA, respectively. CG and G refer to the cyt b562-sfGFP fusion (~40 kDa) and the sfGFP alone (27 kDa) generated through TEV cleavage. The Western blot detection was performed with an antiGFP primary antibody.
Table 1.
Sequence and characteristics of observed cyt b562 TAG replacement variants.
Fig 3.
SPAAC between genetically encoded azide (red sphere) within a protein (blue spheres) and an activate alkyne (dibenzylcyclooctyne;DBCO).
A triazole link is formed between the azide and alkyne groups.
Fig 4.
Production and conjugation of cyt b562 azF-containing variants.
(A) Schematic structure of cyt b562 showing the residues substituted with azF. (B) Expression of cyt b562 variants housed in the pIFtag plasmid in the presence (+azF) or absence (-azF) of azF. Cells successfully expressing functional cyt b562 display a red phenotype. (C) SDS-PAGE analysis of SPAAC conjugation. The top panel shows the Coomassie Blue stained and the bottom is fluorescence imaging of the DBCO-585 moiety. CG, G and C refer to cyt b562-sfGFP fusion, sfGFP and cyt b562, respectively. The lower band is unreacted DBCO-585. For the sake of transparency, the samples Q88 and A91 were run on separate gels to the others and the resulting images electronically linked to the other samples.
Fig 5.
In silico modelling of azF cyt b562 variants.
(A) Models of the A29azF holo cyt b562. The mutated residue, azF29, is coloured in cyan at its carbon atoms. The right hand panel is a close up surface view of the model. (B) Models of the P45azF apo and holo cyt b562 variant. The mutated residue, azF45, is coloured green at its carbon atoms. Haem is represented as spheres. The models were based on the PDB coordinate files 265B (Holo) and 1APU (apo). The figures where generated using PyMol [61].
Fig 6.
In-frame TAG replacement of KGF.
(A) Compatibly of various therapeutic proteins with the E. coli sfGFP fusion screening approach. KGF, keratinocyte growth factor fragment; EPO, ethropoietin; URI, Uricase; ASP+ Asparaginase with N-terminal signal pepetide sequence; ASP- Asparaginase lacking N-terminal signal peptide sequence; SK, Streptokinase. The ‘con’ sample is the control cell sample. The proteins shown are all fused to sfGFP via the TEV digestion sequence. (B) Sequence of observed KGF variants. (C) azF-dependent expression of selected KGF-sfGFP variants. (D) Map of observed mutations on the structure of KGF, including an indication of whether the native residue is surface exposed (blue spheres) or buried (grey spheres) based on surface accessibility.
Fig 7.
Production and SPAAC modification of KGF variants.
(A) Superimposition of the KGF structure on the homologous FGF10-FGFR complex. Yellow and red spheres represent residues contributing to or not involved in the receptor interface. (B) Inherent sfGFP fluorescence of different cellular fractions (top two panels) and SPAAC modification with DBCO-585 of each analysed variant.