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Protein and peptide letters
2024;31 (2): 141-152.

Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

Nana Zhou, Tai An, Yuan Zhang, Guomiao Zhao, Chao Wei, Xuemei Shen, Fan Li, Xiaoyan Wang
Author Information
  1. Nana Zhou: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  2. Tai An: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  3. Yuan Zhang: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  4. Guomiao Zhao: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  5. Chao Wei: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  6. Xuemei Shen: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  7. Fan Li: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
  8. Xiaoyan Wang: Nutrition and Health Research Institute, COFCO Corporation, Beijing 102209, China.
PMID: 38243926 DOI: 10.2174/0109298665276722231212053009

Abstract

BACKGROUND: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue.
OBJECTIVES: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in expression system.
RESULTS: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system.
CONCLUSION: Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the expression system.

Keywords

Photocleavable protein antimicrobial activity. antimicrobial peptides directed evolution expression high throughput screening photocleavage

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Grants

  1. Z191100001119045, No. 20220484146/Beijing Nova Program

MeSH Term

Histatins
Escherichia coli
Recombinant Fusion Proteins
Microbial Sensitivity Tests
Antimicrobial Peptides
Antimicrobial Cationic Peptides
Humans

Chemicals

Histatins
Recombinant Fusion Proteins
Antimicrobial Peptides
Antimicrobial Cationic Peptides
Journal Article

Word Cloud

Created with Highcharts 10.0.0expressionantimicrobialpeptidesmutantHistatin1AntimicrobialproteinR6-2-6-4photocleavagephotocleavablefusionsuccessfullyexpressedsystemPhoClovercometoxicitydegradationhostdirectedevolutionhighthroughputscreeningobtainedsequencepeptidewithingenepurifiedactivityug/mLfermentermg/LHistatin1βLysostaphinPhotocleavableBACKGROUND:AMPspromisingalternativeagentsantibioticsantibioticresistanceproblemsdifficultproducelarge-scaleresearchduetowardshostspeptidasesThereforeheterologousrecombinantalwayschallengingissueOBJECTIVES:lowlevelnewmethodprovided3Methods:higherefficiencyDNAcodingfusedRESULTS:fusingrarelyaffectedMICvalue33basicallyequivalent32chemicallysynthesizedamplification5L-Histatin1improved876also11preparedpowderremainedstablestored4oC4monthswithoutaddition-Defensin105verifiedcertainuniversalityCONCLUSION:-Defensin105mayprovidenovelstrategyexpresspurifyImprovingPhotocleavageEfficiencyProteinPeptideExpression

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