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.
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.
Li
Yifeng
Y
The Structure-Fun Laboratory, Eppley Institute for Research in Cancer and Allied Diseases, Department of Pathology and Microbiology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Li
Xia
X
Wang
Guangshun
G
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D013542:Sweat / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins