Triosephosphate Isomerase by Consensus Design: Dramatic Differences in Physical Properties and Activity of Related Variants

Sullivan, Brandon J.; Durani, Venuka; Magliery, Thomas J.

doi:10.1016/j.jmb.2011.08.001

Cited by 60 publications

(97 citation statements)

References 55 publications

(74 reference statements)

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“…A pET-based plasmid containing the gene encoding the ccTIM protein (Sullivan et al, 2011) with a cleavable 6ÂHis tag (Table 1) was received from Professor Magliery (The Ohio State University, USA). Bacterial cells transformed with this plasmid were grown at 310 K to an OD 600 of $0.8 and then induced for 4 h at 303 K by addition of 0.1 mM isopropyl -d-1-thiogalactopyranoside (IPTG).…”

Section: Expression and Purificationmentioning

confidence: 99%

“…The significance of the database and the methods used for consensus design have been investigated by Magliery and coworkers (Sullivan et al, 2011 for the glycolytic enzyme triosephosphate isomerase (TIM). One of the variants referred to as ccTIM (curated consensus TIM) (Sullivan et al, 2011), which had a consensus mutation at every position (consensus protein), was reported to be almost as active as Saccharomyces cerevisiae TIM despite sharing only 58% sequence identity with it.…”

Section: Introductionmentioning

confidence: 99%

“…One of the variants referred to as ccTIM (curated consensus TIM) (Sullivan et al, 2011), which had a consensus mutation at every position (consensus protein), was reported to be almost as active as Saccharomyces cerevisiae TIM despite sharing only 58% sequence identity with it. The amino-acid sequence of this protein is almost as identical to prokaryotic TIM proteins as to eukaryotic ones.…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, the human and T. maritima sequences are only 42% identical. Further, even though the closest wild-type homologue of ccTIM is from the insect Tenebrio molitor (64% sequence identity; Knobeloch et al, 2010), the database used for its design had significantly greater contribution from bacterial sequences (Sullivan et al, 2011). Hence, ccTIM is neither prokaryotic nor eukaryotic.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, it shares approximately 85% sequence identity with another consensus protein, cTIM (consensus TIM; Sullivan et al, 2011), characterized as a molten globular protein with very little activity. The thermal denaturation profile of ccTIM was reported to be almost identical to that of cTIM.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

et al. 2014

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A bioinformatics-based protein-engineering approach called consensus design led to the construction of a chimeric triosephosphate isomerase (TIM) protein called ccTIM (curated consensus TIM) which is as active as Saccharomyces cerevisiae TIM despite sharing only 58% sequence identity with it. The aminoacid sequence of this novel protein is as identical to native sequences from eukaryotes as to those from prokaryotes and shares some biophysical traits with a molten globular protein. Solving its crystal structure would help in understanding the physical implications of its bioinformatics-based sequence. In this report, the ccTIM protein was successfully crystallized using the microbatch-under-oil method and a full X-ray diffraction data set was collected to 2.2 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C222 1 , with unit-cell parameters a = 107.97, b = 187.21, c = 288.22 Å . Matthews coefficient calculations indicated the presence of six dimers in the asymmetric unit, with an approximate solvent content of 46.2%.

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Section: Expression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

et al. 2014

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Consensus engineering of sucrose phosphorylase: The outcome reflects the sequence input

Aerts

Verhaeghe

Joosten

et al. 2013

Biotech & Bioengineering

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Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison.

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Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor

Case

Hackel

2016

Biotech & Bioengineering

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Protein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged-to-neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches – combinatorial library sorting or consensus design, based on natural homologs or library-sorted mutants – are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd = 11 ±4 nM, Tm = 62 °C, and yield = 4.0 ±0.8 mg/L as compared to 5.3 ±1.7 nM, 71 °C, and 3.5 ±0.3 mg/L for the parental affibody). Extension of consensus design to ten additional mutants exhibited varied performance including a substantially improved mutant (Kd = 6.9 ±1.4 nM, Tm = 71 °C, and 12.7 ±0.9 mg/L yield). Sorting a homolog-based combinatorial library of 7×105 mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd = 1.2 ±0.3 nM, Tm = 69 °C, and 6.0 ±0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (p=0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd = 1.7 ±0.5 nM, Tm = 68 °C, and 7.0 ±0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged-to-neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation.

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Triosephosphate Isomerase by Consensus Design: Dramatic Differences in Physical Properties and Activity of Related Variants

Cited by 60 publications

References 55 publications

Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

Consensus engineering of sucrose phosphorylase: The outcome reflects the sequence input

Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor

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