title: Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris.
abstract: Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants.
authors.affiliation: Thayer School of Engineering, Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhao
authors.forename: Kristina
authors.initials: K
authors.lastname: Blazanovic
authors.forename: Yoonjoo
authors.initials: Y
authors.lastname: Choi
authors.forename: Chris
authors.initials: C
authors.lastname: Bailey-Kellogg
authors.forename: Karl E
authors.initials: KE
authors.lastname: Griswold
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D003062:Codon; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008248:Lysostaphin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010843:Pichia / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D003062:Codon; D005656:Fungal Proteins; D008248:Lysostaphin