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<p>Site-direct mutagenesis provides a basis for functional studies of proteins. Yet, despite many available techniques, mutagenesis of large protein complexes can be tedious and highly challenging, with the replication fidelity and long-range amplification limiting the process. Here, we develop a method for site-directed mutagenesis of large protein complexes based on sub-fragmentation of its coding sequence that generates libraries of shorter sequences, where the DNA polymerase can accurately catalyse long-range elongation. Using the <i>nuo</i> genes encoding for the 0.5 MDa <i>E. coli</i> Complex I, we successfully dissect the 15.1&#xa0;kb long DNA sequence into fragments of 900&#xa0;bp with partially overlapping regions and unique primer sequences, creating a plasmid library that can be amplified by regular DNA polymerases. We showcase the method by sub-cloning the <i>E. coli</i> Complex I sequence into 20 fragments from a large pBAD expression vector (21.3&#xa0;kb). The method shows a high success rate of introduced mutations, and an efficient Gibson assembly of the mutated fragments into the expression vector, enabling the accurate introduction of point-mutations into the large pBAD vector. We suggest that the plasmid sub-fragmentation provides an efficient method for mutagenesis of multi-subunit protein complexes with several homologous subunits, such as the respiratory Complex I, advancing mechanistic studies of large bioenergetics protein complexes.</p>

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Directed mutagenesis of large multi-subunit protein complexes by plasmid sub-fragmentation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月25日

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  • Adel Beghiah,
  • Ville R. I. Kaila

摘要

Site-direct mutagenesis provides a basis for functional studies of proteins. Yet, despite many available techniques, mutagenesis of large protein complexes can be tedious and highly challenging, with the replication fidelity and long-range amplification limiting the process. Here, we develop a method for site-directed mutagenesis of large protein complexes based on sub-fragmentation of its coding sequence that generates libraries of shorter sequences, where the DNA polymerase can accurately catalyse long-range elongation. Using the nuo genes encoding for the 0.5 MDa E. coli Complex I, we successfully dissect the 15.1 kb long DNA sequence into fragments of 900 bp with partially overlapping regions and unique primer sequences, creating a plasmid library that can be amplified by regular DNA polymerases. We showcase the method by sub-cloning the E. coli Complex I sequence into 20 fragments from a large pBAD expression vector (21.3 kb). The method shows a high success rate of introduced mutations, and an efficient Gibson assembly of the mutated fragments into the expression vector, enabling the accurate introduction of point-mutations into the large pBAD vector. We suggest that the plasmid sub-fragmentation provides an efficient method for mutagenesis of multi-subunit protein complexes with several homologous subunits, such as the respiratory Complex I, advancing mechanistic studies of large bioenergetics protein complexes.