1d4 peptide (tetsqvapa)  (GenScript corporation)

Journal: bioRxiv

Article Title: Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

doi: 10.1101/2024.11.21.624670

Figure Lengend Snippet: A , Schematic drawing to describe the construct of Acr InvC-opsin used in this study. In the construct, 36 amino acids of N-terminus in Platynereis c-opsin1 was fused, and C-terminal sequence was removed, and the 1D4 sequence (ETSQVAPA) was added to the opsin. See for detail. B , Absorption spectra of purified Acr InvC-opsin. Spectra in the dark ( black ), after blue illumination ( red ), and after subsequent orange light (>580 nm) illumination ( blue ) are shown. The black and blue spectra are almost superimposed (but see ), indicating that orange light is primarily absorbed by the red-shift photoproduct. In the dark, α max value is 464 nm, and after blue light illumination, the value is shifted to 514 nm. ( Inset ) Absorption spectrum of Acr InvC-opsin in the dark in a wider wavelength range. C , GsX GloSensor assay profiles for Acr InvC-opsin. Blue light induced changes in cAMP biosensor (GloSensor) luminescence in COS-1 cells expressing Gsα ( black ), Gsα/i11 ( red ), Gsα/q11 ( blue ), or Gsα/1211 ( green ) with the InvC-opsin are plotted. Luminescence levels are normalized to the value at the starting point (time = 0 min). Error bars indicate the S.D. values (n = 3). Blue bar shows blue light illumination and the duration of illumination are also indicated. D , Comparison of relative luminescence levels at 20 min (strictly, 20.14 min) in panel C . Error bars indicate the S.D. values (n = 3). The value of “Gsα/i11” is significantly different from the values of “Gsα”, “Gsα/q11”, and “Gsα/1211” ( P < 0.001***). The values of “Gsα/1211” are significantly different from the values of “Gsα” and “Gsα/q11” ( P < 0.001***). The statistical differences were evaluated using Tukey’s test following one-way ANOVA.

Article Snippet: The 1D4 tagged pigments were eluted with buffer A containing 0.45 mg/mL 1D4 peptide (TETSQVAPA) (TOYOBO, Japan).

Techniques: Construct, Sequencing, Purification, Expressing, Comparison

Journal: bioRxiv

Article Title: Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

doi: 10.1101/2024.11.21.624670

Figure Lengend Snippet: A , Amino acid sequence comparison among the reported partial sequence of Acr InvC-opsin (MF145115.1) , addition of the initiation methionine with truncation of C-terminus as well as addition of the 1D4 sequence (“Met-added”), and addition of Platynereis c-opsin1 N-terminal 36 amino acids with truncation of C-terminus as well as addition of the 1D4 sequence (“WT”). Modified amino acids are colored and highlighted in boldface. The position of Ser-94 is also indicated. B , Comparison of expression levels between “Met-added” ( black ) and “WT” ( red ) constructs. Both constructs were expressed in the same number (10 plates) of dishes and purified in parallel. The result indicates that addition of Platynereis c-opsin1 N-terminal 36 amino acids on Acr InvC-opsin increased expression levels in comparison with the simple addition of the initiation methionine.

Article Snippet: The 1D4 tagged pigments were eluted with buffer A containing 0.45 mg/mL 1D4 peptide (TETSQVAPA) (TOYOBO, Japan).

Techniques: Sequencing, Comparison, Modification, Expressing, Construct, Purification