Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli

Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad

doi:10.15171/ijb.1412

Cited by 8 publications

(1 citation statement)

References 22 publications

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“…The data, presented in Figure 3 B, revealed that lower numbers of targets were obtained from genes synthesized artificially ( p = 0.04). Several studies have demonstrated that codon optimization for heterologous production in E. coli can potentially increase recombinant protein expression, especially in sequences of eukaryotic and archaeal origin [ 23 , 24 , 25 , 26 ]. However, other studies revealed that E. coli codon optimization may lead to overexpression of recombinant proteins, thus resulting in a higher tendency to generate insoluble inclusion bodies [ 27 , 28 ].…”

Section: Resultsmentioning

confidence: 99%

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

Cardoso

Brás²,

Costa³

et al. 2022

IJMS

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In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

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