Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment

Liu, Bin; Liu, Shuang; Liu, Jianghai

doi:10.1016/j.nbt.2019.11.004

Cited by 11 publications

(4 citation statements)

References 25 publications

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“…To construct an SH2 variant library, we randomized the eight residues i.e., αA3, BC1,BC2,BC3, βC1, βC3, βD3, βD6 (hereafter referred to position 1 to 8, respectively) (Figure 1a, S2) in the Fyn SH2 domain pTyr binding pocket by Kunkel method 23,24 . Three oligos, which covered the above eight residues in the template SH2 domain in three regions, were designed for mutation (Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Zou

Zhao

et al. 2020

Protein Science

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Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

show abstract

Section: Resultsmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Zou

Zhao

et al. 2020

Protein Science

View full text Add to dashboard Cite

show abstract

“…To construct an SH2 variant library, we randomized the 8 residues(i.e. αA3, BC1, BC2, BC3, βC1, βC3, βD3, βD6 hereafter referred to position 1 to 8, respectively) (Figure 1a and Figure S2) in the Fyn SH2 domain pTyr binding pocket by Kunkel method[23, 24]. Three oligos, which covered the above 8 residues in the template SH2 domain in three regions, were designed for mutation (Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the Identification of novel SH2 superbinders

Zou

Zhao

et al. 2020

Preprint

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Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins, largely antibodies as well as some non-antibody proteins, have undergone directed evolution in vitro in the test tubes in the laboratories around the world, resulted in the numerous protein variants with novel or enhanced functions. In this study, we constructed a Fyn SH2 variant library by randomizing the 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide from MidT antigen led to the identification of SH2 variants with enhanced affinities to the peptide, compared to the wild type SH2, by EC50 assay. Fluorescent polarization (FP) was then applied to quantify the binding affinity of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder (refer to Trm). Biolayer Interferometry (BLI) assay was employed to disclose the kinetics of the binding of these SH2 superbinders, in addition to the wild type SH2, to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. The previously identified SH2 superbinder Trm as well as the V13 and V24 discovered in this study have cross-reactivity with the sulfotyrosine (sTyr) containing peptide while the wild type SH2 does not. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide, implying it binds to the pTyr peptide with a different pattern from the other superbinders. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

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“…[ 55 ] This protocol gives further accessibility to the Kunkel method by providing a description to generate uracil containing templates without the need to use phages. [ 55 ] Other studies to improve the mutation efficiency of Kunkel‐based methods have focused on improving conditions for DNA amplification such as primer design, extension time, and annealing temperature [ 66 ] or the use of phi29 polymerase. [ 67 ]…”

Section: A Established Methods For Sdm and Ssmmentioning

confidence: 99%

Methods for enzyme library creation: Which one will you choose?

2021

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Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.

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Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment

Cited by 11 publications

References 25 publications

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the Identification of novel SH2 superbinders

Methods for enzyme library creation: Which one will you choose?

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