Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris

Appl Environ Microbiol. 2014 May;80(9):2746-53. doi: 10.1128/AEM.03914-13. Epub 2014 Feb 21.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Base Sequence
  • Codon
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Expression*
  • Glycosylation
  • Humans
  • Lysostaphin / chemistry
  • Lysostaphin / metabolism*
  • Lysostaphin / pharmacology
  • Molecular Sequence Data
  • Molecular Structure
  • Pichia / chemistry
  • Pichia / genetics*
  • Pichia / metabolism
  • Protein Engineering
  • Staphylococcal Infections / microbiology
  • Staphylococcus aureus / drug effects


  • Anti-Bacterial Agents
  • Codon
  • Fungal Proteins
  • Lysostaphin

Associated data

  • GENBANK/KF724949