Format

Send to

Choose Destination
Sci Rep. 2015 Dec 17;5:18363. doi: 10.1038/srep18363.

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity.

Author information

1
The Ludwig Center for Cancer Genetics and Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21287, USA.
2
Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.

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.

PMID:
26678960
PMCID:
PMC4683619
DOI:
10.1038/srep18363
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center