Improved protein splicing reaction for low solubility protein fragments without insertion of native extein residues.
Application of trans protein splicing has been limited both by solubility problems and by the insertion of native extein residues (NERs) at the splicing site. Here, we report two simple methods for overcoming these problems and increasing the yield and activity of the spliced product. First, low solubility was alleviated by adding arginine to the reaction buffer and optimizing the splicing reaction condition. The protocol was demonstrated in the context of a Green Fluorescent Protein variant (GFPuv), and the final yield was increased by 1.9-fold compared to control experiments performed under the same conditions but without addition of arginine. Second, the insertion of NERs was overcome by mutating, instead of inserting, a minimal number of residues in the target protein to amino acids required for the splicing reaction. We identified optimal splicing sites that conserve as much as possible the prerequisite NERs. As a result, the GFPuv residues 142-146 (EYNYN) were mutated to the reportedly minimal required NERs, EYCFN. GFPs spliced using this strategy had no NERs insertion and a fluorescence activity six times stronger than a control GFPuv with five NERs inserted at the splicing site (residue 145/6). In principle, the present protocol (Sw/oNI) can be applied to any target protein, even when no sequence similarity to NERs is present, though it will introduce up to five mutations at the splicing site.
Kamioka
Tetsuya
T
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Tawa
Minako
M
Sohya
Shihori
S
Yamazaki
Toshio
T
Kuroda
Yutaka
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004258:DNA Polymerase III / Q000235:genetics; D047669:Exteins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000235:genetics
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III