Production of soluble and active microbial transglutaminase in Escherichia coli for site-specific antibody drug conjugation.
Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Mathias
http://orcid.org/0000-0003-4034-9090
M
Rickert
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Pavel
P
Strop
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Victor
V
Lui
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Jody
J
Melton-Witt
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Santiago Esteban
SE
Farias
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Davide
D
Foletti
David
D
Shelton
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Jaume
J
Pons
Rinat Laboratories, Pfizer, Inc, 230 East Grand Avenue, South San Francisco, California, 94080, USA.
Arvind
A
Rajpal
D000595:Amino Acid Sequence; D001483:Base Sequence; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D018796:Immunoconjugates / Q000378:metabolism*; D007218:Industrial Microbiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012995:Solubility; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D018796:Immunoconjugates; D011503:Transglutaminases
E. coli; S. mobarensis; antibody drug conjugation; cloning; microbial transglutaminase; protein purification; soluble expression