High-Level Production of Bacteriotoxic Phospholipase A1 in Bacterial Host Pseudomonas fluorescens via ABC Transporter-Mediated Secretion and Inducible Expression

Park, Jiyeon; Eom, Gyeong Tae; Oh, Joon Young; Park, Ji Hyun; Kim, Sun Chang; Song, Jae Kwang; Ahn, Jung Hoon

doi:10.3390/microorganisms8020239

Cited by 12 publications

(7 citation statements)

References 49 publications

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“…To determine the effect of temperature on refolded PLB1 activity, PLB1 and yolk lecithin mixtures were incubated for 15 min (pH 7.0) at different temperatures, ranging from 20 to 80 °C. PLB1 activity was measured by pH-stat technique as previously described (Park et al 2020). The amount of free fatty acids released from a lecithin solution can be quantified by adding 0.01 M NaOH to maintain a constant pH of 7.0 using an automatic pH-stat (Metrohm, Switzerland).…”

Section: Effects Of Ph and Temperature On Recombinant Plb1 Activitymentioning

confidence: 99%

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli

Zhao¹,

Zhou²,

Wang

et al. 2020

3 Biotech

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Candida albicans is an important fungal pathogen that causes a wide variety of human infections, ranging from mucocutaneous infections to life-threatening systemic infections. Phospholipase B1 (PLB1) has been reported to be directly responsible for C. albicans pathogenicity and is likely to be involved in the early steps of host invasion. Therefore, PLB1 could be a potential marker for diagnosis of C. albicans infection. In this study, PLB1 was expressed using an Escherichia coli expression system. Recombinant PLB1 is found in inclusion bodies and constitutes up to 38.4% of total insoluble protein. After refolding in a GSH/GSSG redox system, GST-tagged PLB1 was purified by GST-sepharose 4B affinity chromatography and then cleaved with thrombin to remove the GST-tag. The recombinant PLB1 was further purified by anion-exchange chromatography and reverse phase HPLC. The final yield of purified PLB1 was approximately 15.6 mg from 100 mL of bacterial cell culture, and its concentration was 784 μg/μL. The recombinant PLB1 could form a white precipitation zone on egg yolk agar plate, suggesting its phospholipase activity. Moreover, the maximum activity of PLB1 was 68 IU/mg at pH 6.0, 37 °C. Therefore, recombinant PLB1 has potential application in structural analytical studies, or diagnosis of C. albicans infection.

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Section: Effects Of Ph and Temperature On Recombinant Plb1 Activitymentioning

confidence: 99%

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli

Zhao¹,

Zhou²,

Wang

et al. 2020

3 Biotech

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“…In the last decades, several PLA 1 genes from microorganisms had been cloned and expressed and the most frequently studied PLA 1 genes were from Serratia sp. strains [9][10][11][12]. However, PLA 1 secreted by Serratia sp.…”

Section: Introductionmentioning

confidence: 96%

Heterologous expression, refolding, and characterization of a calcium-independent phospholipase A1 from Streptomyces albidoflavus

Cheng

Guo

Liang

et al. 2022

Syst Microbiol and Biomanuf

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Phospholipase A 1 (PLA 1 ) is a member of the hydrolase family with applications in various fields, especially in the food industry. A calcium-independent PLA 1 from Streptomyces albidoflavus was expressed in E. coli BL21 (DE3) in this study. The results indicated that the soluble expression of PLA 1 was at low level, which was possibly due to the toxicity of PLA 1 to the host. In contrast, the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium. Furthermore, the renaturation of PLA 1 was achieved through a direct dilution method, yielding 29.6 U/mL PLA 1 activity after 16 h of renaturation at 4 °C. For improving the efficiency of the dilution refolding process, a continuous refolding strategy was established, and 155 U/mL PLA 1 activity was derived from the continuous refolding process. With soybean lecithin as the substrate, the specific activity of purified renatured PLA 1 was 1380 U/mg and the optimal temperature and pH was found to be 60 °C and 6.5. In addition, the renatured PLA 1 was observed with better activity towards phosphatidyl inositol, whilst lipase activity was detected when the catalyzing temperature was below 55 °C. Overall, this study provides a possible solution to obtain calcium-independent PLA 1 with high yield by heterologous expression in E. coli and hence to promote its further application in the field of food industry.

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“…A wide variety of proteins are secreted through this oligomeric secretion channel to play their biological roles. Due to the simple structure of the system, T1SSs have been widely applied in biomedical engineering applications ( Schwarz et al, 2012 ; Ryu et al, 2015 ; Park et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

T1SEstacker: A Tri-Layer Stacking Model Effectively Predicts Bacterial Type 1 Secreted Proteins Based on C-Terminal Non-repeats-in-Toxin-Motif Sequence Features

et al. 2022

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Type 1 secretion systems play important roles in pathogenicity of Gram-negative bacteria. However, the substrate secretion mechanism remains largely unknown. In this research, we observed the sequence features of repeats-in-toxin (RTX) proteins, a major class of type 1 secreted effectors (T1SEs). We found striking non-RTX-motif amino acid composition patterns at the C termini, most typically exemplified by the enriched “[FLI][VAI]” at the most C-terminal two positions. Machine-learning models, including deep-learning ones, were trained using these sequence-based non-RTX-motif features and further combined into a tri-layer stacking model, T1SEstacker, which predicted the RTX proteins accurately, with a fivefold cross-validated sensitivity of ∼0.89 at the specificity of ∼0.94. Besides substrates with RTX motifs, T1SEstacker can also well distinguish non-RTX-motif T1SEs, further suggesting their potential existence of common secretion signals. T1SEstacker was applied to predict T1SEs from the genomes of representative Salmonella strains, and we found that both the number and composition of T1SEs varied among strains. The number of T1SEs is estimated to reach 100 or more in each strain, much larger than what we expected. In summary, we made comprehensive sequence analysis on the type 1 secreted RTX proteins, identified common sequence-based features at the C termini, and developed a stacking model that can predict type 1 secreted proteins accurately.

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High-Level Production of Bacteriotoxic Phospholipase A1 in Bacterial Host Pseudomonas fluorescens via ABC Transporter-Mediated Secretion and Inducible Expression

Cited by 12 publications

References 49 publications

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli

Heterologous expression, refolding, and characterization of a calcium-independent phospholipase A1 from Streptomyces albidoflavus

T1SEstacker: A Tri-Layer Stacking Model Effectively Predicts Bacterial Type 1 Secreted Proteins Based on C-Terminal Non-repeats-in-Toxin-Motif Sequence Features

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