Protein Technology Core

Division Of Basic Sciences - Nci

Investigators

Abstract

The Protein Purification and Protein Engineering Cores supported and critically contributed to the research programs of several laboratories at NCI's Center for Structural Biology from October 2021 to June 2022. Specifically, these efforts centered on providing biochemical protein reagents such as site-specific proteolytic and glycolytic enzymes for the cleavage of covalently fused protein tags during the purification of proteins of interest. In additional efforts, the staff produced proteins such as engineered streptavidin and nanobodies for the preparation of customized affinity matrices as well as enzymes for molecular biology applications. The work of research laboratories focused on membrane proteins was supported by the Cores through regular production of high-quality proteins required for the generation of nanodisc assemblies. The Cores also contributed to individual research programs supporting the work of postdoctoral and postbaccalaureate fellows by establishing protocols for the production of their target proteins. Core staff also assisted researchers with tissue culture by maintaining stocks of human cell lines to produce challenging proteins and multiprotein complexes. The Protein Technologies Core (PTC) was established and started its operations as a replacement for the Protein Purification and Protein Engineering Cores on July 1, 2022. The PTC will incorporate and expand the protein production capabilities by retaining experienced personnel who are all highly experienced in protein production, purification, biophysical characterization, and tissue culture. An immediate priority for the PTC is to provide the personnel with additional training and professional development to modernize and refresh their skills. The equipment for protein purification is undergoing significant modernization with the latest state-of-the-art platforms, including microfluidic capabilities already procured for the facility. The production of proteins is an essential requisite for structural biology applications. However, the ability to produce milligrams of pure and biochemically stable proteins and multiprotein complexes can pose significant challenges for individual labs. The PTC will aim to support researchers in this task by employing semi-automated, multi-pronged approaches at each workflow stage, from construct design to cloning, expression, and purification, through the characterization of most protein targets, including membrane proteins and multi-protein complexes. At the outset, the PTC will implement a digital lab notebook to trace and visualize the exact lab workflow. Digital record management will significantly improve productivity and protocol development through collaboration between the PTC staff and research labs. In addition, digitization of PTC records will ensure compliance with the storage and inventory of valuable new biochemical reagents to safeguard valuable intellectual property and ensure compliance with institutional policies. An early and essential strategic goal for the PTC is to establish a pipeline for producing and characterizing binder proteins. Binders are a broad characterization applied to proteins such as single-chain camelid antibodies (nanobodies) and their derivatives that interact with high affinity with a chosen target protein. Binders have broad applications in structural biology as tools for stabilizing and purifying proteins and protein complexes. One important application is in single-particle cryoelectron microscopy of membrane proteins, where binders help find favorable orientations of particles in vitreous ice to enable structure determination. The PTC will establish the cloning, production, purification, and characterization of binders to several important targets from libraries derived from the directed evolution of nanobody scaffolds. The application of binders developed and discovered by the PTC will be further extended to super-resolution imaging and crosslinking mass spectrometry to study protein structure, functions, and protein-protein interactions. In addition to technology and research support, the PTC will be committed to providing training and support for researchers at all levels with particular needs with protein production and characterization.