Abstract
N α-Acetylation is one of the most common protein modifications in eukaryotes but a rare event in prokaryotes. Some endogenously N α-acetylated proteins in eukaryotes are frequently reported not to be acetylated or only very partially when expressed in recombinant Escherichia coli. Thymosin α1 (Tα1), an N α-acetylated peptide of 28 amino acids, displays a powerful general immunostimulating activity. Here, we revealed that a fusion protein of thymosin α1 and L12 is partly N α-acetylated in E. coli. Through deletion of some N α-acetyltransferases by Red recombination, we found that, when rimJ is disrupted, the fusion protein is completely unacetylated. The relationship of rimJ and N α-acetylation of Tα1 was further investigated by gene rescue and in vitro modification. Our results demonstrate that N α-acetylation of recombinant Tα1-fused protein in E. coli is catalyzed by RimJ and that fully acetylated Tα1 can be obtained by co-expressing with RimJ. This is the first description that an ectopic protein acetylation in bacterial expression systems is catalyzed by RimJ, a known prokaryotic N α-acetyltransferase.
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Acknowledgments
We are very grateful to the unknown international reviewer for his/her careful rewriting of this paper. We also thank Ms. ** Li for help in the Q-TOF MS analysis and sequencing. This work was supported by the National Natural Science Foundation of China under grant no. 30780049 to H. F.
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Fig. S1-1
The nucleotide sequence and amino acid sequence of the fusion protein Tα1-L12 (DOC 12 kb)
Fig. S1-2
Expression and purification of the recombinant Tα1-L12 in Escherichia coli (DOC 68 kb)
Fig. S1-3
Expression and purification of the recombinant RimJ in Escherichia coli (DOC 41 kb)
Fig. S1-4
Q-TOF mass spectrum of purified recombinant Tα1-L12 expressed in JM109(DE3) (A), JM109(DE3) rimI − (B), JM109(DE3) rimJ − (C), JM109(DE3) rimL − (D), JM109(DE3) yjaB − (E), and co-expressed with pACYC-rimJ in JM109(DE3) rimJ − (F) (DOC 97 kb)
Table S1-1
The primers for deletion of E. coli NAT’s gene by Red recombination (13 kb)
Supplementary material-S2
The identification of the acetylating position of recombinant Tα1-L12. Fig. S2-1 Detection of the double-charged ions [M+2H]2+ of the non-modified N-terminal 1-14 peptide (m/z = 712.78) and the modified one (m/z = 733.78) by LC-MS (note that residue numbering excluding the first methionine because it is cleaved in the mature protein). Fig. S2-2 The sequencing analysis of the double-charged ions [M+2H]2+ of the non-modified N-terminal 1–14 peptide (m/z = 712.78) (note that residue numbering excludes the first methionine since it is cleaved in the mature protein) by nanoESI Q-TOF MS/MS. Fig. S2-3 The sequencing analysis of the double-charged ions [M+2H]2+ of the modified N-terminal 1–14 peptide (m/z = 733.78) (note that residue numbering excludes the first methionine since it is cleaved in the mature protein) by nanoESI Q-TOF MS/MS for sequencing; the acetylation position was located at N-terminal serine residue of recombinant Tα1-L12 (DOC 13 kb)
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Fang, H., Zhang, X., Shen, L. et al. RimJ is responsible for N α-acetylation of thymosin α1 in Escherichia coli . Appl Microbiol Biotechnol 84, 99–104 (2009). https://doi.org/10.1007/s00253-009-1994-8
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DOI: https://doi.org/10.1007/s00253-009-1994-8