Facile construction of a random protein domain insertion library using an engineered transposon

Shah, Vandan; Pierre, Brennal; Kim, Jin Ryoun

doi:10.1016/j.ab.2012.09.030

Cited by 7 publications

(4 citation statements)

References 45 publications

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“…This commentary presents our recent development on combinatorial approaches for the construction of insertional fusion libraries and circular permutation libraries using engineered Mu transposons. 22,23 Transposons for random insertional fusion of protein domains Insertional protein fusion is an advanced engineering approach to generate novel proteins with integrated functions. Three critical parameters that can determine the success of functional integration between fused proteins are the insertion location within the host protein, 24,25 the inter-domain linker length 26,27 and the inter-domain linker composition.…”

Section: Introductionmentioning

confidence: 99%

“…22 A schematic of the construction of random domain insertion libraries using the MuST transposon is shown in Fig. 1A.…”

Section: Introductionmentioning

confidence: 99%

“…1B). 22 Second, placement of the restriction sites at the ends required minimal mutations outside or on the near ends of the MuA transposase recognition binding sites. The minimal sequence change was desired to maintain the transposition efficiency as compared to the wildtype Mu transposon.…”

Section: Introductionmentioning

confidence: 99%

“…Also, the ratio of having correctly oriented to inversely oriented insertions of a guest DNA was about 1:1, though slight variation was also observed from run to run. 22,23 Transposons for random circular permutation of proteins Circular permutation is a protein engineering approach that connects the original N-and C-termini of proteins through a linker and, instead, introduces new termini elsewhere in the sequence. 38 As more than 50% of single domain proteins have their N-and C-termini proximal, 39 circular permutation could be an effective tool to generate diversity in the linear order of amino acid sequences.…”

Section: Introductionmentioning

confidence: 99%

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Transposon for protein engineering

Shah

Kim

2016

Mobile Genetic Elements

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Protein insertional fusion and circular permutation are 2 promising protein engineering techniques for creating integrated functionalities and sequence diversity of a protein, respectively. Finding insertion locations for protein insertional fusion and new termini for circular permutation through a rational approach is not always straightforward, especially, for proteins without detailed structural knowledge. On the contrary, a combinatorial approach facilitates a comprehensive search to evaluate all potential insertion sites and new termini locations. Conventional methods used to create random insertional fusion libraries generate sub-optimal inter-domain linker length and composition between fused proteins. There are also methods available for construction of random circular permutation libraries. However, these methods too, impose many drawbacks, such as significant sequence modification at the new termini of circular permutants and additionally, require re-design of transposons for tailored expression of circular permutants. Furthermore, these conventional methods employ relatively inefficient blunt-end ligation during library construction. In this commentary, we present a concise overview and key findings of engineered Mu transposons, which have recently been developed in our group as a facile and efficient tool to alleviate limitations realized from conventional methods and to construct high quality libraries for random insertional fusion and random circular permutation.

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Section: Introductionmentioning

confidence: 99%

“…22 A schematic of the construction of random domain insertion libraries using the MuST transposon is shown in Fig. 1A.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Transposon for protein engineering

Shah

Kim

2016

Mobile Genetic Elements

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Engineering Allostery into Proteins

Gorman

D'Amico

Winston

et al. 2019

Advances in Experimental Medicine and Biology

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Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH

Shah

Charlton

Kim

2017

Appl Biochem Biotechnol

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High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165, the corresponding unfused BCX mutant, BCX, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.

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Facile construction of a random protein domain insertion library using an engineered transposon

Cited by 7 publications

References 45 publications

Transposon for protein engineering

Transposon for protein engineering

Engineering Allostery into Proteins

Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH

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