In this article, we focus on the folding trajectories of two mini proteins: BBA5 (Protein Data Bank ID) [ 9] and GSGS (orBeta3s) [ 10, 11].
For instance, the aforementioned Anaplasma includes two sequenced species, A. phagocytophilum HZ (contains 33.39% mini-proteins) and A. marginale str.
We first analyzed the overall length distribution of mini-proteins and described the main characteristics of the each phylum.
As a result, we found many new groups of conserved mini-proteins and obtained 28 groups of proteins conforming to the above conditions.
It is plausible that even subtle changes in the environment may become a selective pressure for mini-proteins, and the differences among Clostridium are the result of multifactor influence.
They include (i) how to identify potentially more mini-proteins in various genomes; (ii) how to confirm the functions of identified mini-proteins; (iii) what are the biological functions of the mini-proteins; and (iv) what are the driving forces for the evolution of the mini-proteins.
Individual samples (15 μl) were loaded into the wells of a 10% acrylamide/bis gel (29 1), containing 7% glycerol, and 1X TBE buffer and casted in mini protein tetra vertical chamber (Bio-Rad).
After a 24-h incubation, cells were washed with 1xPBS and then lysed in culture dish with shaking for 15 min at 4°C with ice-cold lysis buffer (150 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet-P40, 0.5% natrium deoxycholate, 1 tablet Complete Mini protein inhibitor mixture/10 ml (Roche Applied Science), and 0.7 µg/ml pepstatin).
All centrifugation steps were carried out at 1,200× g for 1 min at 4°C. 1. Washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet-P40, 0.5% natrium deoxycholate, 1 tablet Complete Mini protein inhibitor mixture/10 ml (Roche Applied Science) and 0.7 µg/ml pepstatin).
All western blots were performed on Invitrogen Mini Cell and Bio-Rad Mini Protein Apparatus using the Bio-Rad 034BR power pack.
The quality of the NFAP2 fractions was checked by SDS-PAGE (Novex™ 18 % Tris Glycine Mini Protein Gels, 1.0 mm, 10-well; Thermo Fisher Scientific, Waltham, MA, USA).
