ABSTRACT
The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.
Subject(s)
Antibodies, Anti-Idiotypic/isolation & purification , Antibodies, Monoclonal/isolation & purification , Progesterone/immunology , Secretory Leukocyte Peptidase Inhibitor/isolation & purification , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Bacterial Proteins/chemistry , Chromatography, Affinity/methods , Mice , Milk/chemistry , Secretory Leukocyte Peptidase Inhibitor/chemistry , Staphylococcal Protein A/chemistryABSTRACT
The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
Subject(s)
Pichia/chemistry , Pichia/metabolism , Secretory Leukocyte Peptidase Inhibitor/biosynthesis , Secretory Leukocyte Peptidase Inhibitor/chemistry , Cell Culture Techniques , Fermentation , Glycosylation , Humans , Pichia/genetics , Protein Folding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Secretory Leukocyte Peptidase Inhibitor/genetics , Secretory Leukocyte Peptidase Inhibitor/isolation & purification , Transfection , Trypsin/metabolismABSTRACT
BACKGROUND: The aim of this study was to separate bactericidal proteins from healthy female cervical mucus. METHODS: Cervical mucus was collected and dissolved in 1% acetic acid. The antimicrobial activity of acid-soluble extracts was detected by gel overlay assay against Escherichia coli ATCC 43827. The extracts showed considerable amount of antibacterial activity with a clearly visible band. The bactericidal band was purified by reverse-phase high performance liquid chromatography and the antibacterial activity of the eluate was examined using radial diffusion assay. RESULTS: Two antimicrobial proteins were purified and were further characterized by Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis, N-terminal sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The proteins were identified as high-mobility group nucleosomal-binding domain 2 (HMG N2) and secretory leukocyte peptidase inhibitor (SLPI). SLPI is an antimicrobial peptide already known in the cervical mucus while HMG N2 in the cervical mucus had not been previously reported. The expression of HMG N2 mRNA was detected in Hela cells and cervical epithelial cells by RT-PCR. Slit hybridization showed abundant amounts of the HMG N2 protein in the cervical mucus. CONCLUSIONS: These results suggest that the expression of HMG N2 and SLPI in the healthy female cervical mucus may be relevant to their immune surveillance and defense against potential pathogens in human reproductive system.
