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, 2.1mg 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 microM, 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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Li Y,Li X,Wang Gdoi
10.1016/j.pep.2007.04.023subject
Has Abstractpub_date
2007-10-01 00:00:00pages
395-405issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(07)00126-Xjournal_volume
55pub_type
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