A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation

Yifeng Li, Xia Li, He Li, Oksana Lockridge, Guangshun Wang

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (∼95 kDa) at pH ∼7, allowing a rapid and clean separation from the carrier thioredoxin (∼14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 °C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.

Original languageEnglish (US)
Pages (from-to)157-165
Number of pages9
JournalProtein Expression and Purification
Volume54
Issue number1
DOIs
StatePublished - Jul 1 2007

Fingerprint

formic acid
Agglomeration
Peptides
Fusion reactions
Affinity chromatography
Phosphatidylglycerols
Thioredoxins
Size exclusion chromatography
High Pressure Liquid Chromatography
Micelles
Thrombin
Isotopes
Purification
Carrier Proteins
Feasibility Studies
Proteins
Metals
Affinity Chromatography
Innate Immunity
Nuclear magnetic resonance

Keywords

  • Antimicrobial peptides
  • Cathelicidins
  • Chemical cleavage
  • Enzyme cleavage
  • Escherichia coli
  • FPLC
  • HPLC
  • LL-37
  • NMR
  • Protein aggregation

ASJC Scopus subject areas

  • Biotechnology

Cite this

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title = "A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation",
abstract = "The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (∼95 kDa) at pH ∼7, allowing a rapid and clean separation from the carrier thioredoxin (∼14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50{\%} formic acid at 50 °C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53{\%}. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.",
keywords = "Antimicrobial peptides, Cathelicidins, Chemical cleavage, Enzyme cleavage, Escherichia coli, FPLC, HPLC, LL-37, NMR, Protein aggregation",
author = "Yifeng Li and Xia Li and He Li and Oksana Lockridge and Guangshun Wang",
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T1 - A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation

AU - Li, Yifeng

AU - Li, Xia

AU - Li, He

AU - Lockridge, Oksana

AU - Wang, Guangshun

PY - 2007/7/1

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N2 - The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (∼95 kDa) at pH ∼7, allowing a rapid and clean separation from the carrier thioredoxin (∼14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 °C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.

AB - The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (∼95 kDa) at pH ∼7, allowing a rapid and clean separation from the carrier thioredoxin (∼14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 °C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.

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