Expression and purification of the native C‐amidated antimicrobial peptide maculatin 1.1

Zhu, Shiying; Weber, Daniel K.; Separovic, Frances; Sani, Marc‐Antoine

doi:10.1002/psc.3330

Cited by 5 publications

(5 citation statements)

References 45 publications

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“…Like melittin, many antimicrobial peptides have an amidated C-terminus (Table S1). 1 The C-terminal amide increases these peptides' α-helical propensity 2 and promotes interactions with cellular membranes, which facilitates pore formation, thus conferring their membrane-lytic properties. 3 C-Terminal amidation is therefore crucial to study the native structure and dynamics of such peptides.…”

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confidence: 99%

“…This has been achieved by (1) harvesting the peptides from eukaryotic cells or organisms that do the posttranslational modification; 4 (2) solid-phase peptide synthesis (SPPS) with a resin that results in a C-terminal amide after cleavage; 5 (3) recombinant expression in bacterial hosts followed by the enzymatic conversion of a C-terminal glycine into an amide; 6 or (4) recombinant expression of intein fusion proteins followed by cleavage in the presence of high concentrations of dithiothreitol and ammonium bicarbonate. 1,7 Heteronuclear nuclear magnetic resonance (NMR) spectroscopy experiments require isotopic enrichment of 15 N, 13 C, and/or 2 H within the peptide. Site-specific labeling of 15 N and 13 C with SPPS quickly becomes cost-prohibitive with more than a handful of labeled residues.…”

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confidence: 99%

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Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

Gelenter

Bax

2023

J. Am. Chem. Soc.

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Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled ( 2 H, 13 C, and 15 N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3−K23.

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confidence: 99%

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confidence: 99%

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

Gelenter

Bax

2023

J. Am. Chem. Soc.

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“…That the entire system (containing Bsa BI blunt restriction enzyme site) can be transferred to any vector, we confirmed by the construction of additional pQE_Ek expression vector (Figure 1). Based on the latest results in the development of new improved expression vectors, we can conclude that the improvement of expression vectors will most likely go in the direction of reducing the toxicity of expressed AMPs (sandwich tag vector construction) and those contributing to AMP modification to increase stability and functionality (Lamer et al, 2022; Zhu et al, 2021). The improvements we have presented in this study can be combined with other improvements (most are compatible) in the construction of new expression vectors with multiple tags (sandwich tag) and with enzymatic modifications and processing.…”

Section: Discussionmentioning

confidence: 99%

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

Gardijan

Miljković

Obradović

et al. 2022

Journal of Applied Microbiology

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The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results:To improve the pMAL expression vector, we introduced the His 6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His 6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His 6 tagged enterokinase.In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His 6 and His 6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human βdefensin. Conclusions: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides.Significance and Impact of the Study: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory. K E Y W O R D Scomplete removal of tags and proteinase, His 6 -enterokinase cleavage site, improved pMAL and pQE vectors, native antimicrobial peptides, two-step chromatography

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“…Even for relatively short AMPs, 15 N- and 13 C-labeled samples for NMR experiments are difficult to obtain via chemical synthesis. Recombinant expression is an effective solution to these problems. , …”

Section: Introductionmentioning

confidence: 99%

“…Recombinant expression is an effective solution to these problems. 17,18 The traditional recombinant expression system using Escherichia coli (E. coli) can at times be ineffective in expressing AMPs. Various proteases in the cell can pose a threat to short peptides with a simple structure.…”

Section: Introductionmentioning

confidence: 99%

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues

Kato

Kumeta

et al. 2022

ACS Omega

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Cecropin P1 (CP1) isolated from a large roundworm Ascaris suum , which is found in pig intestines, has been extensively studied as a model antimicrobial peptide (AMP). However, despite being a model AMP, its antibacterial mechanism is not well understood, particularly the function of its C-terminus. By using an Escherichia coli overexpression system with calmodulin as a fusion partner, we succeeded in the mass expression of recombinant peptides, avoiding toxicity to the host and degradation of CP1. The structure of the recombinant 15 N- and 13 C-labeled CP1 and its C-terminus truncated analogue in dodecylphosphocholine (DPC) micelles was determined by NMR. In this membrane-mimetic environment, CP1 formed an α-helix for almost its entire length, except for a short region at the C-terminus, and there was no evidence of a hinge, which is considered important for the expression of activity in other cecropins. Several NMR analyses showed that the entire length of CP1 was protected from water by micelles. Since the loss of the C-terminus of the analogue had little effect on the NMR structure or its interaction with the micelle, we investigated another role of the C-terminus of CP1 in its antimicrobial activity. The results showed that the C-terminal region affected the DNA-binding capacity of CP1, and this mechanism of action was also newly suggested that it contributed to the antimicrobial activity of CP1.

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Expression and purification of the native C‐amidated antimicrobial peptide maculatin 1.1

Cited by 5 publications

References 45 publications

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues

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