Protein evolution by codon-based random deletions

Osuna, Joel

doi:10.1093/nar/gnh135

Cited by 21 publications

(25 citation statements)

References 33 publications

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“…9a, which is published as supporting information on the PNAS web site). Several tolerated short deletions and insertions in the ⍀-loop have been described, some of which result in altered substrate specificity (17,18).…”

Section: Resultsmentioning

confidence: 99%

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

202

246

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We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 ␤-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of ␤-lactam hydrolysis. Some of these MBP-BLA switches were effectively ''on-off'' in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth͞no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 ؋ 10 6 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a ''sucrose-binding protein,'' illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.allostery ͉ ␤-lactamase ͉ maltose binding protein R egulation of protein activity is fundamental to cellular function.One of the mechanisms a cell uses to modulate the level of protein activity is regulation of the amount of a protein present in a cell. Accordingly, many strategies for engineering control of cellular protein activity have focused on modulation of protein production and degradation, often through small-moleculedependent switches that regulate transcription, translation, localization, degradation, or protein splicing (1) or through the engineering of artificial gene-regulatory networks (2). The key strength of many of these approaches is that they are easily generalized. For example, a switch that regulates transcription of one gene can easily be adapted to regulate transcription of an arbitrary gene. However, the significant limitation of these approaches is the slow dynamics stemming from the indirect nature of the regulation (i.e., protein activity is regulated by controlling the amount of protein and not by regulating the protein's specific activity directly). In addition, modulation is only feasible in the context of the cell and cannot be easily transferred to an in vitro setting.A more satisfying approach is to modulate the protein activity directly, but this presents a considerable design problem. Inhibitors, if they can be found, can only can be used to down-regulate activity, and such a strategy suffers from the fact that one is not free to choose the signal that modulates the activity: The signal must be an inhibitor of the protein. One clever way around this limitation is to engineer the inhibitor such that a third molecule can regulate the inhibitor's affinity for the regulated protein, as was demonstrated for RNA aptamer inhibitors that could be regulated by an organic small molecule (3). Natural allosteric proteins solve this problem by having spatially distinct regulatory and active sites. Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has ...

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Section: Resultsmentioning

confidence: 99%

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

202

246

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“…Although the three other motifs SXXK, SDN, and K(S/T)G are conserved, the absence of Glu 166 suggested that these proteins are rather PBPs than ␤-lactamases. Although substitutions in the ⍀-loop can occur naturally, giving rise to extended spectrum lactamases (25), insertions or deletions were mainly genetically engineered in this region (26,27). Only one example of a natural duplication in the ⍀-loop of a class A ␤-lactamase has been reported (28).…”

Section: Resultsmentioning

confidence: 99%

A New Family of Cyanobacterial Penicillin-binding Proteins

Urbach

Fastrez

Soumillion

2008

Journal of Biological Chemistry

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It is largely accepted that serine ␤-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with ␤-lactam antibiotics, such as penicillins. On the other hand, ␤-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and ␤-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a ␤-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A ␤-lactamases. Interestingly, the ⍀-loop, which, in the ␤-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate ␤-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter ⍀-loop.D-Alanyl-D-alanine peptidases are enzymes involved in the synthesis of the peptidoglycan, the bacterial cell wall constituent that is responsible for the cell shape and resistance to osmotic pressure (1). These enzymes catalyze transpeptidation and carboxypeptidation reactions, thus controlling the peptidoglycan synthesis and cross-linking. As a result of the acylation of their catalytic serine, these enzymes form stable acylenzymes with ␤-lactam antibiotics, such as penicillins, the stability of these complexes being at the origin of the antibiotic effect. Hence, DD-peptidases are also called penicillin-binding proteins or PBPs 2 (2, 3). PBPs are divided into two main groups: the low M r PBPs are monofunctional catalytic entities (4), and the high M r PBPs comprise an additional N-terminal domain (5). To counteract ␤-lactam antibiotics, some bacteria produce ␤-lactamases (6 -8), which are enzymes able to hydrolyze penicillins up to 10 8 times faster than PBPs. According to their primary structure, serine ␤-lactamases can be divided into three classes: A, C, and D (class B constituting the group of metalloenzymes). The penicillin-binding module of DD-peptidases and ␤-lactamases share a similar three-dimensional structure composed of two domains, an all-␣-domain and an ␣/␤-domain, the catalytic site being at the interface (7). Three common essential motifs line up the active site, following Ambler numbering (9), S 70 XXK containing the active nucleophile serine, (S/Y) 130 XN on a loop in the all-␣-domain and (K/R) 234 (S/T)G on a ␤-sheet forming the opposite wall of the catalytic cavity. Regarding ␤-lactamases, the rapi...

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“…Recent developments in protein engineering have proposed random amino acid deletions as a good alternative to classic amino acid substitution mutagenesis to expand protein sequence space [Osuna et al, 2004;Raghunathan et al, 2012]. Here we presented evidence that codon deletion is a feasible approach with which to enhance SD accessibility and protein expression.…”

Section: Discussionmentioning

confidence: 80%

“…In our previous work, we used an oligonucleotide library assembled by the Codon-Based Random Deletion (COBARDE) mutagenesis approach [Osuna et al, 2004] and successfully isolated two functional deletion mutants, sgGFPΔI128 and sgGFPΔD129, which remained robustly fluorescent under direct visualization with sunlight. In fact, E. coli cells expressing sgGFPΔD129 at 37 ° C were slightly less fluorescent than were those expressing the parental protein sgGFP and were more fluorescent at 30 ° C. A similar result was obtained by Liu et al [2015] for several internal deletion mutants of GFPuv, the relative fluorescence of which was improved at a lower temperature.…”

Section: Discussionmentioning

confidence: 99%

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Rodríguez-Mejía¹,

Roldán‐Salgado²,

Osuna³

et al. 2016

Microb Physiol

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Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

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Protein evolution by codon-based random deletions

Cited by 21 publications

References 33 publications

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

A New Family of Cyanobacterial Penicillin-binding Proteins

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

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