Molecular diversity in engineered protein libraries

Barakat, Nora H.; Love, John J.

doi:10.1016/j.cbpa.2007.05.014

Cited by 5 publications

(8 citation statements)

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“…Exploring protein sequence space with combinatorial libraries of amino acid sequences has become an important strategy towards understanding the relationship between protein sequence, structure and function [1, 2]. While numerous approaches introduce sequence diversity, the likelihood of identifying/developing proteins with altered/novel functions is increased when the library of genes encodes for folded and soluble structures [2, 3].…”

Section: Introductionmentioning

confidence: 99%

“…While numerous approaches introduce sequence diversity, the likelihood of identifying/developing proteins with altered/novel functions is increased when the library of genes encodes for folded and soluble structures [2, 3]. Thus using high-quality library approaches that generate large collections of overexpressed, soluble proteins are desirable for this long-term goal.…”

Section: Introductionmentioning

confidence: 99%

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Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Bradley¹,

Bricken²,

Randle³

2011

Protein Expression and Purification

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Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Bradley¹,

Bricken²,

Randle³

2011

Protein Expression and Purification

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“…To optimize the folding, we screened libraries of XFP0.7 as C-terminal fusions to oligomeric Aβ42 that is known to render bright FP with poor folding weakly fluorescent. 9,10 After four rounds of random mutagenesis and screening, we identified a bright fluorescent variant, dubbed mIFP663, that possesses 13 mutations: N6D, V29A, R120G, V127T, T128V, M133L, Q203L, V230M, M259I, T262S, V296L, S309G, and V314E (Figure S3).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“… a Excitation peak. b Emission peak. c Extinction coefficient (M –1 cm –1 ). d Quantum yield (%) e Normalized molecular brightness (EC × QY) to that of mIFP663. f Values from the original papers. ,,,, g Values from this study. …”

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confidence: 98%

A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein–Protein Interaction

Liu

Deng

et al. 2022

ACS Sens.

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Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M −1 cm −1 ) and a decent quantum yield (19.4%), and high cellular brightness that is 3−6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein−protein interactions in living cells.

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“…The difficulty of the search increases with the complexity, but reduction of the library complexity potentially sacrifices—removes—the best recognition sequences even before the search is undertaken. Even with the best library construction techniques, the limit to actual sequence complexity is on the order of 10 8 to 10 12 unique sequences 12, 13. In a fully randomized 8‐mer peptide, there are 208 (or 2.56 × 10 10 ) possible sequence combinations, thus making it unlikely that even the best constructed library can cover much of sequence space.…”

Section: Introductionmentioning

confidence: 99%

De novoidentification of binding sequences for antibody replacement molecules

2009

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A new in silico method has been developed that automatically identifies peptide sequences that can bind to targets of known three-dimensional structure. The method is potentially faster and more economical than traditional methods of raising antibodies by means of hybridomas or biopanning technology. The current algorithm creates an initial peptide library that is either completely random or that is constrained by the user. This library represents only a small fraction of possible sequence space and the peptides are created with a specified torsional geometry. The library is used as input to any number of available molecular docking programs and the library is docked and scored. The final rank ordering is then used to create a new library by constraining that library to the sequence conservation pattern deduced from the top N-scoring peptides in the first round. Successive rounds of screening, scoring, and new library creation ultimately results in the system converging to a final solution set of peptides. To test the method, a family of novel peptides that can bind to, and inhibit the enzyme Deoxyribonuclease I has been discovered. The peptides inhibit the enzyme either alone or when placed into a protein backbone structure as has been confirmed experimentally.

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Molecular diversity in engineered protein libraries

Cited by 5 publications

References 50 publications

Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein–Protein Interaction

De novoidentification of binding sequences for antibody replacement molecules

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