title: PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity.
abstract: Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Surojit
authors.initials: S
authors.lastname: Sur
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Qiao
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Anja
authors.initials: A
authors.lastname: Fries
authors.affiliation: Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
authors.forename: Robert N
authors.initials: RN
authors.lastname: O'Meally
authors.affiliation: Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Kinzler
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Bert
authors.initials: B
authors.lastname: Vogelstein
authors.affiliation: The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
authors.forename: Shibin
authors.initials: S
authors.lastname: Zhou
mesh_terms: D000588:Amines / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D005260:Female; D006207:Half-Life; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D011092:Polyethylene Glycols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013388:Succinimides / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000737:chemistry* / Q000493:pharmacokinetics
chemical_list: D000588:Amines; D013388:Succinimides; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; C001426:N-hydroxysuccinimide