title: Purification and characterization of human procolipase expressed in yeast cells.
abstract: We report the successful, efficient, and large-scale expression of recombinant human procolipase in yeast. Using the full-length cDNA of human procolipase, constructs were made using either the native human procolipase signal peptide sequence or the signal peptide sequence of yeast. These constructs were used to transform yeast cells, and expression was followed. Only minimal expression was seen with the procolipase using the native human signal peptide. Robust secretion of the procolipase occurred when the yeast signal peptide was exchanged for the native signal peptide. Expression yielded more than 30 mg/liter. The recombinant protein was purified from the medium by immunoaffinity chromatography. The highly purified procolipase was free of proteolytic degradation and displayed activity and binding characteristics that were indistinguishable from those of tissue-purified human pancreatic colipase. Expression in yeast cells provides a useful tool for expressing intact, unprocessed recombinant wild-type and mutated procolipase.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Cordle
authors.forename: M E
authors.initials: ME
authors.lastname: Lowe
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D003089:Colipases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003089:Colipases; D004792:Enzyme Precursors; D011498:Protein Precursors; D011994:Recombinant Proteins; C037107:procolipase