Next Article in Journal
Genome Editing Using Mammalian Haploid Cells
Next Article in Special Issue
High-Throughput Screening in Protein Engineering: Recent Advances and Future Perspectives
Previous Article in Journal
Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target
Previous Article in Special Issue
Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research
Article Menu
Issue 10 (October) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2015, 16(10), 23587-23603; doi:10.3390/ijms161023587

Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

1
Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA
2
Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, ON M5G1L7, Canada
Current address: Meso Scale Diagnostics, 1601 Research Blvd., Rockville, MD 20850, USA.
Current address: Integrated DNA Technologies, 8180 McCormick Blvd., Skokie, IL 60076, USA.
*
Author to whom correspondence should be addressed.
Academic Editor: Qiang “Shawn” Chen
Received: 21 August 2015 / Revised: 18 September 2015 / Accepted: 23 September 2015 / Published: 30 September 2015
(This article belongs to the Special Issue Protein Engineering)
View Full-Text   |   Download PDF [1449 KB, uploaded 30 September 2015]   |  

Abstract

Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. View Full-Text
Keywords: affinity maturation; affinity selection; error-prone PCR; FN3 monobody; loop shuffling; Kunkel mutagenesis; megaprimer; off-rate selection; phage-display; secondary library affinity maturation; affinity selection; error-prone PCR; FN3 monobody; loop shuffling; Kunkel mutagenesis; megaprimer; off-rate selection; phage-display; secondary library
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Huang, R.; Gorman, K.T.; Vinci, C.R.; Dobrovetsky, E.; Gräslund, S.; Kay, B.K. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step. Int. J. Mol. Sci. 2015, 16, 23587-23603.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top