On-resin cleavage of bacterially expressed fusion proteins for purification of active recombinant peptides SK-29, KR-20, LL-29, and LL-23 from human sweat or skin

Yifeng Li, Xia Li, Guangshun Wang

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1 mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 μM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.

Original languageEnglish (US)
Pages (from-to)395-405
Number of pages11
JournalProtein Expression and Purification
Volume55
Issue number2
DOIs
StatePublished - Oct 1 2007

Fingerprint

Sweat
Purification
Skin
Fusion reactions
Resins
Peptides
Peptide Fragments
Proteins
formic acid
High Pressure Liquid Chromatography
Agglomeration
Escherichia coli K12
Microbial Sensitivity Tests
Centrifugation
Cloning
Thrombin
Culture Media
Organism Cloning
Membrane Proteins
Escherichia coli

Keywords

  • Antimicrobial peptides
  • Double cleavage
  • Escherichia coli
  • HPLC
  • LL-37
  • NMR
  • On-resin cleavage
  • Peptide aggregation
  • Target identification

ASJC Scopus subject areas

  • Biotechnology

Cite this

@article{fb0be9ba8a6f4b91b48efde6cd8c00fc,
title = "On-resin cleavage of bacterially expressed fusion proteins for purification of active recombinant peptides SK-29, KR-20, LL-29, and LL-23 from human sweat or skin",
abstract = "Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1 mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 μM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.",
keywords = "Antimicrobial peptides, Double cleavage, Escherichia coli, HPLC, LL-37, NMR, On-resin cleavage, Peptide aggregation, Target identification",
author = "Yifeng Li and Xia Li and Guangshun Wang",
year = "2007",
month = "10",
day = "1",
doi = "10.1016/j.pep.2007.04.023",
language = "English (US)",
volume = "55",
pages = "395--405",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - On-resin cleavage of bacterially expressed fusion proteins for purification of active recombinant peptides SK-29, KR-20, LL-29, and LL-23 from human sweat or skin

AU - Li, Yifeng

AU - Li, Xia

AU - Wang, Guangshun

PY - 2007/10/1

Y1 - 2007/10/1

N2 - Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1 mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 μM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.

AB - Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1 mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 μM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.

KW - Antimicrobial peptides

KW - Double cleavage

KW - Escherichia coli

KW - HPLC

KW - LL-37

KW - NMR

KW - On-resin cleavage

KW - Peptide aggregation

KW - Target identification

UR - http://www.scopus.com/inward/record.url?scp=34548487515&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548487515&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2007.04.023

DO - 10.1016/j.pep.2007.04.023

M3 - Article

VL - 55

SP - 395

EP - 405

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -