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.

journal_name

Appl Environ Microbiol

authors

Zhao H,Blazanovic K,Choi Y,Bailey-Kellogg C,Griswold KE

doi

10.1128/AEM.03914-13

subject

Has Abstract

pub_date

2014-05-01 00:00:00

pages

2746-53

issue

9

eissn

0099-2240

issn

1098-5336

pii

AEM.03914-13

journal_volume

80

pub_type

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