Fig. 1: BBP reconstituted in β-carotene synthesizing E. coli cells is equivalent to the native yellow protein extracted from locust cuticle.

a The photograph of two locust specimens used to extract yellow protein from the cuticles (panel b) and to collect Raman spectra (panel c). b The appearance of the centrifuged extracts into the SEC buffer obtained from the two locust specimens. c Raman spectra collected from the cuticles of the two locust specimens as compared with the spectra of β-carotene in n-hexane and in BBP. The main Raman bands are marked and the discussed maxima of the peaks are indicated (in cm⁻¹). d SEC profiles of the recombinant and native BBP (Superdex 200 Increase 5/150; 0.45 mL min⁻¹). Numbers indicate the apparent M_w values in kDa obtained from column calibration. Vo - void volume. e Absorbance spectra of the recombinant and native BBP as compared with the spectrum of BmCBP complexed with ZEA. The main maxima positions are indicated in nm. f Amino acid sequence of the final BBP construct corresponding to the mature form devoid of the N-terminal signal peptide. Cys residues are red, Trp residue is light blue, Lys residues are orange, the C-terminal tail is pink. Below the sequence the prediction of the intrinsic disorder propensity is found. g SDS-PAGE of the fractions obtained upon BBP isolation after cell lysis in buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) without or then with 2 M urea. Super and pellet are fractions obtained after sonication and centrifugation, of which the former was loaded onto HisTrap 5 mL column (Flow - flowthrough fraction), washed with buffer A (wash A) and then eluted by 500 mM imidazole (fractions El1, El2, El3 were collected consecutively). M designates protein markers with known M_w (indicated in kDa on the right). Arrow on the left indicates the apparent M_w of BBP with the His₆ tag. Barbed arrows indicate the effect of urea on BBP yield. h SEC profiles at 450 nm of BBP obtained by loading aliquots of BBP eluates from IMAC, pre-dialyzed against buffer A, for regular lysis and extraction (no urea) or extraction in the presence of 2 M urea (Superdex 200 Increase 10/300 column; 0.8 mL min⁻¹). The absorbance spectra for the peaks 1 and 2 registered throughout the elution profiles are shown in insert. Typical SDS-PAGE analysis of the fractions collected during the profiles is shown in the insert below. The appearance of the recombinant BBP sample loaded on the SEC column is shown in the insert. i Far-UV circular dichroism spectrum of the purified BBP as compared with the circular dichroism spectrum calculated by PDBMD2CD web server⁴⁷ for an Alphafold 2-derived model of BBP (colored by secondary structure element types) with or without the C-terminus.