Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins.
Inteins are protein segments embedded in frame within a precursor sequence that catalyze a self-excision reaction and ligate the flanking sequences with a standard peptide bond. Split inteins are expressed as two separate polypeptide fragments and trans-splice upon subunit association. Split inteins have found use in biotechnology applications but their use in postsynthetic domain assembly in vivo has been limited to the ligation of two protein domains. Alternatively, they have been used to splice three domains and fragments in vitro. To further develop split intein-based applications in vivo, we have designed a cell-based assay for the postsynthetic splicing of three protein domains using orthogonal split inteins. Using naturally and artificially split inteins, NpuDnaE and SspDnaB, we show that a multidomain protein of 128 kDa can be assembled in Escherichia coli from individually expressed domains. In the current system, the main bottleneck in achieving high yield of tandem trans-spliced product appears to be the limited solubility of the SspDnaB precursors. Optimizing protein solubility should be important to achieve efficient combinatorial synthesis of protein domains in the cell.
Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, 14260, NY, USA.
Daniel
D
Demonte
Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, 14260, NY, USA.
Naiyi
N
Li
Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, 14260, NY, USA. sjpark6@buffalo.edu.
Sheldon
S
Park
D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D047668:Inteins / Q000502:physiology*; D019154:Protein Splicing / Q000502:physiology*; D017434:Protein Structure, Tertiary
C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; C506730:dnaB protein, E coli; D053488:DnaB Helicases
Domain assembly; NpuDnaE; Protein ligation; Protein trans-splicing; Split intein; SspDnaB