Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system.
Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (approximately 10 microg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.
Kanno
Takuya
T
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan.
Kitano
Michiko
M
Kato
Rika
R
Omori
Akira
A
Endo
Yaeta
Y
Tozawa
Yuzuru
Y
D000595:Amino Acid Sequence; D002474:Cell-Free System; D017931:DNA Primers; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000502:physiology*; D014908:Triticum / Q000502:physiology*
D017931:DNA Primers; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D011994:Recombinant Proteins; D008715:Methionine; D005982:Glutathione Transferase