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DOU Tonglu, CHEN Hao, CAO Jin, CHEN Junsheng, ZHU Jianwei. Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area[J]. Journal of China Pharmaceutical University, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515
Citation: DOU Tonglu, CHEN Hao, CAO Jin, CHEN Junsheng, ZHU Jianwei. Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area[J]. Journal of China Pharmaceutical University, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515
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Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area

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    • Abstract
  • Intein is a functional protein that mediates self-cleavage from precursor protein and simultaneously connects the exteins on both sides of the intein via peptide bonds. Among all kinds of inteins, split intein has a wide range of applications in the field of antibody ligation. However, since the naturally split intein specifically recognizes the first three amino acid sequences “cysteine, phenylalanine, and asparagine”(CFN)of the extein, exogenous amino acids are inevitably introduced after the protein splicing reaction. In this study, the amino acid sequence “cysteine, aspartic acid, and lysine”(CDK)of the antibody hinge region was substituted for “CFN” as the recognition site for the intein and the split intein Npu DnaE was mutated into Npu*GEP DnaE. The results showed that the mutant could recognize “CDK” and the intein splicing reaction could successfully take place. The factors affecting the intein splicing reaction platform, such as pH, temperature and the concentration of NaCl and DTT were investigated in this study. The results showed that the splicing reaction of the mutant performed well, which indicated its potential usefulness in bispecific antibody assembly. In conclusion, the problem of introducing foreign amino acids was alleviated, the broadness of intein substrate was further expanded, and further technical support for the application of intein to the antibody assembly was provided.
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    • References (19)
  • [1]
    Lennon CW,Belfort M.Inteins[J].Curr Biol,2017,27(6):R204-R206.
    [2]
    Carvajal-Vallejos P,Pallissé R,Mootz HD,et al.Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources[J].J Biol Chem,2012,287(34):28686-28696.
    [3]
    Xu YR,Zhang L,Ma BY,et al.Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE[J].Enzyme Microb Technol,2018,118:6-12.
    [4]
    Stevens AJ,Sekar G,Gramespacher JA,et al.An atypical mechanism of split intein molecular recognition and folding[J].J Am Chem Soc,2018,140(37):11791-11799.
    [5]
    Gramespacher JA,Stevens AJ,Thompson RE,et al.Improved protein splicing using embedded split inteins[J].Protein Sci,2018,27(3):614-619.
    [6]
    Stevens AJ,Sekar G,Shah NH,et al.A promiscuous split intein with expanded protein engineering applications[J].Proc Natl Acad Sci U S A,2017,114(32):8538-8543.
    [7]
    Iwai H,Züger S,Jin J,et al.Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme[J].FEBS Lett,2006,580(7):1853-1858.
    [8]
    Li YF.Split-inteins and their bioapplications[J].Biotechnol Lett,2015,37(11):2121-2137.
    [9]
    Mills KV,Johnson MA,Perler FB.Protein splicing:how inteins escape from precursor proteins[J].J Biol Chem,2014,289(21):14498-14505.
    [10]
    Shi SW,Chen HH,Jiang H,et al.A novel self-cleavable tag Zbasic-ΔI-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli[J].Appl Microbiol Biotechnol,2017,101(3):1133-1142.
    [11]
    Wang J,Han L,Chen JS,et al.Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells[J].Int Immunopharmacol,2019,66:288-295.
    [12]
    Scott CP,Abel-Santos E,Wall M,et al.Production of cyclic peptides and proteins in vivo[J].Proc Natl Acad Sci U S A,1999,96(24):13638-13643.
    [13]
    Giriat I,Muir TW.Protein semi-synthesis in living cells[J].J Am Chem Soc,2003,125(24):7180-7181.
    [14]
    Han L,Chen JS,Ding K,et al.Efficient generation of bispecific IgG antibodies by split intein mediated protein trans-splicing system[J].Sci Rep,2017,7(1):8360.
    [15]
    Han L,Zong HF,Zhou YX,et al.Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies[J].Methods,2019,154:32-37.
    [16]
    Shah NH,Eryilmaz E,Cowburn D,et al.Extein residues play an intimate role in the rate-limiting step of protein trans-splicing[J].J Am Chem Soc,2013,135(15):5839-5847.
    [17]
    Cheriyan M,Pedamallu CS,Tori K,et al.Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues[J].J Biol Chem,2013,288(9):6202-6211.
    [18]
    Mills KV,Perler FB.The mechanism of intein-mediated protein splicing:variations on a theme[J].Protein Pept Lett,2005,12(8):751-755.
    [19]
    Johnson MA,Southworth MW,Herrmann T,et al.NMR structure of a KlbA intein precursor from Methanococcus jannaschii[J].Protein Sci,2007,16(7):1316-1328.
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