Construction of a random circular permutation library using an engineered transposon

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

doi:10.1016/j.ab.2014.12.011

Cited by 8 publications

(9 citation statements)

References 42 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%

“…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%

“…23 The design of MuRCP transposon involves attachment of 2 restriction sites at the 5 0 and 3 0 ends of the Mu transposon, respectively, which not only facilitate sticky-end ligation in library construction, but also reduces modifications at the new termini of random circular permutants. Additionally, the MuRCP transposon requires no permanent built-in elements for expression; instead, expression can be externally modulated by the selection of an appropriate cloning vector.…”

Section: Introductionmentioning

confidence: 99%

“…It should, however, be noted that the transposition efficiency of the MuRCP transposons was lower by >»6-fold than the MuST transposon. 23 This differential transposon efficiency might be due to the difference in the number of necessary nucleotide mutations in order to incorporate the restriction sites at the ends of transposons. For the MuRCP transposon, inclusion of the NdeI site at the 5 0 end requires 4 nucleotide mutations as compared to 2 nucleotide mutations for the BclI site included in the MuST trasnsposon.…”

Section: Introductionmentioning

confidence: 99%

“…Any restriction site other than SpeI may be selected and incorporated at both ends of the target gene, which would code a different peptidyl linker. 23 …”

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%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Any restriction site other than SpeI may be selected and incorporated at both ends of the target gene, which would code a different peptidyl linker. 23 …”

Section: Introductionmentioning

confidence: 99%

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