High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris

Tang, Wei; Li, Zhezhe; Li, Chunhua; Yu, Xianhong; Wang, Fei; Wan, Xin; Wang, Yaping; Ma, Lixin

doi:10.1016/j.pep.2016.02.009

Cited by 11 publications

(3 citation statements)

References 30 publications

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“…Combining these data with past results ( 30 ), we conclude that LapA is active against ≥10 substrates. Most substrates had nonpolar, amino acid targets, but, given its cleavage of aspartate, lysine, serine, and tyrosine aminopeptides, LapA has rather broad activity ( 37 , 38 ). Typically of a member of the M28 family ( 39 ), LapA was inhibited by bestatin ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas

White

Gunderson

Tyson

et al. 2018

mBio

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Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC. A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii. A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein.

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Section: Resultsmentioning

confidence: 99%

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas

White

Gunderson

Tyson

et al. 2018

mBio

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“…According to the results reported by Bao et al., only 34% of nanobody was lost . A yield of 79.61% was showed by the group of Tang who purified aminopeptidase . Thus, higher recovery yield might be obtained if the procedure of IMAC purification could be further optimized.…”

Section: Resultsmentioning

confidence: 97%

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli

Song

Jia

2017

Biotech and App Biochem

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Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics.

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“…subtilis str. BSP1 [34]. To assess thermostability, the enzyme solutions were pre-incubated for 3 h at 30 • C, 40 • C, 50 • C, 60 • C, 70 • C and 80 • C, respectively, and then the enzyme activity was determined at 58 • C and the relative enzyme was calculated.…”

Section: Effects Of Temperature and Ph On Purified Aminopeptidasementioning

confidence: 99%

Expression, Characterisation, Homology Modelling and Molecular Docking of a Novel M17 Family Leucyl-Aminopeptidase from Bacillus cereus CZ

Liu,

Cui

2023

IJMS

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Leucyl-aminopeptidase (LAP), an important metallopeptidase, hydrolyses amino acid residues from the N-terminus of polypeptides and proteins, acting preferentially on the peptide bond formed by N-terminus leucine. A new leucyl-aminopeptidase was found in Bacillus cereus CZ. Its gene (bclap) contained a 1485 bp ORF encoding 494 amino acids with a molecular weight of 54 kDa. The bcLAP protein was successfully expressed in E. coli BL21(DE3). Optimal activity is obtained at pH 9.0 and 58 °C. The bcLAP displays a moderate thermostability and an alkaline pH adaptation range. Enzymatic activity is dramatically enhanced by Ni2+. EDTA significantly inhibits the enzymatic activity, and bestatin and SDS also show strong inhibition. The three-dimensional model of bcLAP monomer and homohexamer is simulated byPHYRE2 server and SWISS-MODEL server. The docking of bestatin, Leu-Trp, Asp-Trp and Ala-Ala-Gly to bcLAP is performed using AutoDock4.2.5, respectively. Molecular docking results show that the residues Lys260, Asp265, Lys272, Asp283, Asp342, Glu344, Arg346, Gly372 and His437 are involved in the hydrogen bonding with the ligands and zinc ions. There may be two nucleophilic catalytic mechanisms in bcLAP, one involving His 437 or Arg346 and the other involving His437 and Arg346. The bcLAP can hydrolyse the peptide bonds in Leu-Trp, Asp-Trp and Ala-Ala-Gly.

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High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris

Cited by 11 publications

References 30 publications

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli

Expression, Characterisation, Homology Modelling and Molecular Docking of a Novel M17 Family Leucyl-Aminopeptidase from Bacillus cereus CZ

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