Isolation and Functional Properties of Highly-Purified N-terminal Domain of Human NaPi2b by Scalable “Resin Overload” Technique

Authors

Daria Savenkova
Irina Makarenko
Daria Nedorezova
Ramziya Kiyamova
Mikhail Bogdanov

Language

English

Publication Date

8-1-2025

Journal

Analytical Biochemistry

DOI

10.1016/j.ab.2025.115875

PMID

40254166

PMCID

PMC12077926

PubMedCentral® Posted Date

10-1-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

Despite the enthusiasm and advances in the purification of native and engineered full-length membrane proteins, little attention has been paid to their fragments which could serve as attractive inspiration for function, regulation, or targeting of full-length membrane protein with therapeutic antibodies (Abs). Production of recombinant fragments of "therapeutic" membrane proteins for early-stage discovery research requires their purification to near homogeneity. It is important not only for the production of biotherapeutic antibodies but also for structural and functional studies of competitive protein-Abs, protein-protein, and lipid-protein interactions which heavily rely on the purity and quality of the isolated protein fragment of interest. The development of novel strategies for simple but still highly efficient protein purification remains a one of main research focus in the biotechnology and biomedicine because conventional purification approaches require complex manipulation steps and are timely and costly. Here, we would like to introduce a simple and rapid protein purification strategy for the human NaPi2b N-terminal (NT) sequence recombinantly expressed in a bacterial host at a laboratory scale. We demonstrate that "resin overload" e.g. the conditions when loading exceeds dynamic binding capacity can be counterintuitively but intelligently utilized to isolate highly purified protein fragments and prevent non-specific low-affinity binding of contaminant endogenous host proteins. The results showed that this method allowed us to achieve the highest purity while maintaining both immunogenic (recognition by Abs) and functional (phosphorylation) properties of the NaPi2b NT sequence. Although adaptations are required on a case-to-case basis, we believe this work can inspire other researchers working with the purification of protein and protein fragments to apply this proof-of-principle in a scalable manner.

Keywords

Humans, Recombinant Proteins, Protein Domains, Chromatography, Affinity, Escherichia coli, Purification, NaPi2b, Membrane protein, N-terminal domains, Peptide

Published Open-Access

yes

Recommended Citation

Savenkova, Daria; Makarenko, Irina; Nedorezova, Daria; et al., "Isolation and Functional Properties of Highly-Purified N-terminal Domain of Human NaPi2b by Scalable “Resin Overload” Technique" (2025). Faculty, Staff and Student Publications. 5372.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/5372