Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Inteins are protein subsequences that catalyze their own excision and ligate flanking protein sequences.
• Split inteins, expressed as separate fragments, can trans-splice upon association, offering biotechnological applications.
• Current in vivo applications of split inteins are limited to ligating two protein domains.

Purpose of the Study:

• To design and validate a cell-based assay for the postsynthetic splicing of three protein domains in vivo.
• To expand the utility of split inteins for assembling complex, multidomain proteins within a cellular environment.
• To investigate the feasibility of combinatorial protein synthesis using split intein technology.

Main Methods:

• Utilized orthogonal split inteins, specifically NpuDnaE and SspDnaB, for tandem trans-splicing.
• Developed a cell-based assay in Escherichia coli for monitoring and quantifying the assembly of a 128 kDa multidomain protein.
• Expressed individual protein domains separately before initiating the trans-splicing reaction.

Main Results:

• Successfully demonstrated the in vivo assembly of a 128 kDa multidomain protein from three individually expressed domains using split inteins.
• Identified limited precursor solubility (SspDnaB) as a key bottleneck affecting the yield of the tandem trans-spliced product.
• Established a proof-of-concept for postsynthetic assembly of complex protein structures within a bacterial host.

Conclusions:

• The developed cell-based assay enables efficient in vivo postsynthetic splicing of three protein domains.
• Improving the solubility of split intein precursors is crucial for optimizing the yield of assembled multidomain proteins.
• This approach holds promise for advancing combinatorial protein synthesis and engineering applications.