Expression of recombinant human neutrophil Cathepsin G in Pichia pastoris

Abstract

Cathespin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG has dual specificity for chymotrypsin‐like and tryspin‐like substrates. CatG is a poorly understood enzyme and is currently only commercially available as mature enzyme purified from human sources. The yeast Pichia pastoris is being used to express CatG to study its dual specificity and its C‐terminal processing. The full length (C‐terminus present) human CatG amino acid sequence was modified to remove one glycosylation site and eight dibasic sites to avoid potential cleavage by yeast kexin protease. The construct was engineered to have an N‐terminal 6‐His‐cytochrome B5 (CytB5) heme binding fusion domain linked to the modified human CatG by an enterokinase cleavage site for activation. The amino acid sequence was used to generate a codon‐optimized gene that was placed in the pPICzα secretion vector. After transforming Pichia pastoris strain X‐33, 48 Zeocin‐resistant clones were screened for relative levels of CatG activity. Recombinant CatG has been partially purified from fermentation media by nickel affinity chromatography and its activity has been confirmed by assays using synthetic substrates. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.