Rapid Golden Gate assembly of exons from genomic DNA for protein expression in
            <i>Escherichia coli</i>
            and
            <i>Pichia Pastoris</i>

Cheng, Junhao; Wu, Mingkun; Zhong, Ren; Si, Daoyuan; Meng, Geng; Zhang, Rijun; Zhang, Yueping

doi:10.2144/btn-2021-0039

Cited by 3 publications

(5 citation statements)

References 12 publications

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“…In our previous work, we constructed an effective plasmid construction strategy, using Golden Gate cloning technology to clone the target gene into the pET-Amp plasmid [ 37 ]. The diagrammatic sketch of the construction of the recombinant expression plasmid was shown in Figure S1 .…”

Section: Resultsmentioning

confidence: 99%

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

et al. 2021

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CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

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Section: Resultsmentioning

confidence: 99%

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

et al. 2021

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“…Previously, an effective strategy for the rapid construction of target genes into the pPICZαA-Amp plasmid using the golden gate technology was developed [ 35 ]. The schematic diagram of the recombinant plasmid construction is shown in Figure 1 .…”

Section: Resultsmentioning

confidence: 99%

“…In the present study, we hypothesized that the combination of CM4 [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ], LL37 [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ], and TP5 [ 1 , 2 , 3 , 4 , 5 ] may have amended LPS neutralizing and anti-inflammatory activity with the least cytotoxic and hemolytic effects. Consequently, we synthesized and expressed the hybrid peptide CLP fused with HSA in methylotrophic yeast and studied its natural activities.…”

Section: Introductionmentioning

confidence: 99%

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization

et al. 2023

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The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

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“…In a more recent study, Cheng et al used the Golden Gate cloning method to develop a versatile and easy way of assembling eukaryotic gene exons into both prokaryotic and eukaryotic plasmids in a one-step reaction [64]. Thus, this new approach enables researches to rapidly identify the optimal expression host for the production of specific proteins, overcoming some disadvantages of traditional methods to obtain intron-free eukaryotic genes (e.g.…”

Section: Pichia Pastorismentioning

confidence: 99%

“…Thus, this new approach enables researches to rapidly identify the optimal expression host for the production of specific proteins, overcoming some disadvantages of traditional methods to obtain intron-free eukaryotic genes (e.g. whole-gene synthesis or reverse transcription methods) which are time-consuming, expensive and complicated to operate [64].…”

Section: Pichia Pastorismentioning

confidence: 99%

Upgrading Non-Conventional Yeasts into Valuable Biofactories

Castillo-Mendieta¹,

Arias²,

Gonzales-Zubiate³

2023

Biomedical Engineering

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The use of synthetic biology on yeasts has enhanced the production of commercially relevant chemicals, from biofuels to recombinant therapeutic proteins, to name just a few. Despite most of these advances had already been studied and described in Saccharomyces cerevisiae, during the last years the attention has turned to the use of alternative expression systems with a higher yield and quality such as non-conventional yeasts. Recently, there has been an increase in studies about non-conventional yeasts due to advantages based on their natural capacity to tolerate harsh conditions or the wide range of carbon sources they need during the generation of specific products. This chapter, therefore, aims to describe the current status of the most used non-conventional yeasts in metabolite production as well as the engineering behind them in order to optimize or regulate protein expression: Pichia pastoris, Kluyveromyces marxianus, Kluyveromyces lactis and Yarrowia lipolytica.

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Rapid Golden Gate assembly of exons from genomic DNA for protein expression in Escherichia coli and Pichia Pastoris

Cited by 3 publications

References 12 publications

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization

Upgrading Non-Conventional Yeasts into Valuable Biofactories

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