High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

Cheng, Kuang-Ting; Wu, Chih-Lung; Yip, Bak-Sau; Yu, Huiyuan; Cheng, Hsi-Tsung; Chih, Ya-Han; Cheng, Jya‐Wei

doi:10.3390/molecules23040800

Cited by 18 publications

(10 citation statements)

References 30 publications

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“…Small-molecule AMPs were often expressed as fusion proteins [22,27]. A disadvantage of fusion protein expression for AMPs is that fusion proteins need to be processed in vitro, by proteases or chemicals, to release AMPs.…”

Section: Discussionmentioning

confidence: 99%

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

Fan

Lin

et al. 2020

Bioengineered

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The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

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Section: Discussionmentioning

confidence: 99%

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

Fan

Lin

et al. 2020

Bioengineered

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“…To obtain 15 N-, 13 C-labeled peptide for three-dimensional NMR analysis, P-113 was expressed in Escherichia coli BL21 (DE3) cells grown in M9 minimal media containing 15 N-labeled ammonium chloride (1 g/L) and 13 C-labeled glucose (2 g/L). Peptides were purified according to our previous study [32]. All NMR samples were prepared in SD broth containing 10% D 2 O.…”

Section: Nuclear Magnetic Resonance Spectroscopymentioning

confidence: 99%

The Interactions between the Antimicrobial Peptide P-113 and Living Candida albicans Cells Shed Light on Mechanisms of Antifungal Activity and Resistance

Cheng

Yip

et al. 2020

IJMS

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In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi’s resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.

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“…Several efforts have been made to identify fragments of histatin 5 with pharmaceutical application and have yielded promising results. An example in this regard is the 12-amino acid peptide P113, which was evaluated in phase I and phase II clinical studies as pharmaceutical agent to fight oral candidiasis ( Woong et al, 2008 ; Cheng et al, 2018 ; Browne et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Antimicrobial Peptides: A New Hope in Biomedical and Pharmaceutical Fields

Moretta

Scieuzo

Petrone

et al. 2021

Front. Cell. Infect. Microbiol.

316

198

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Antibiotics are essential drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. Antibiotic resistance is a serious challenge and has led to the need for new alternative molecules less prone to bacterial resistance. Antimicrobial peptides (AMPs) have aroused great interest as potential next-generation antibiotics, since they are bioactive small proteins, naturally produced by all living organisms, and representing the first line of defense against fungi, viruses and bacteria. AMPs are commonly classified according to their sources, which are represented by microorganisms, plants and animals, as well as to their secondary structure, their biosynthesis and their mechanism of action. They find application in different fields such as agriculture, food industry and medicine, on which we focused our attention in this review. Particularly, we examined AMP potential applicability in wound healing, skin infections and metabolic syndrome, considering their ability to act as potential Angiotensin-Converting Enzyme I and pancreatic lipase inhibitory peptides as well as antioxidant peptides. Moreover, we argued about the pharmacokinetic and pharmacodynamic approaches to develop new antibiotics, the drug development strategies and the formulation approaches which need to be taken into account in developing clinically suitable AMP applications.

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High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

Cited by 18 publications

References 30 publications

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

The Interactions between the Antimicrobial Peptide P-113 and Living Candida albicans Cells Shed Light on Mechanisms of Antifungal Activity and Resistance

Antimicrobial Peptides: A New Hope in Biomedical and Pharmaceutical Fields

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