# Membrane Protein Expression, Purification

> **NIH NIH P50** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $136,262

## Abstract

Membrane proteins (MPs) constitute a special set of problems concerning expression in a functional form, and
purification to a pure, homogeneous and stable entity suitable for structural approaches using both cryo-EM
(Core 3) and X-ray methods (Core 6). Less than 1% of the entries in the protein structure database are
membrane proteins and most of these are bacterial proteins, reflecting the much greater difficulties of suitable
expression, purification and crystallization of membrane proteins, and particularly eukaryotic membrane
proteins. Among the accessory proteins in HIV is the membrane protein Vpu. Vpu interacts with several host
soluble and membrane proteins including two well-characterized targets for degradation via ubiquitination
pathways, these being the receptor for HIV, CD4, and BST-2 (tetherin). Mutation of the gene, selection of
constructs, and expression of Vpu, and formation of complexes with both soluble and membrane proteins are
therefore a high priority. Vpu also forms a pentameric viroporin structure that we prepare for structure analysis
and determination in Project 3.
The accessory protein Nef, together with the clathrin adaptor AP-2 and other factors, form an interaction
network with host 10-crossing integral membrane proteins SERINC3, or SERINC5 to target these restriction
factors for destruction. Expression of SERINC3 and 5 are a priority for the goals of Project 4, which are to
define this interaction network at atomic resolution. The core will screen a variety of species and constructs to
obtain the quantity and quality of protein needed for structure analysis. We have developed a general method
to prioritize, express, purify and crystallize integral membrane proteins. We will express, purify,
thermodynamically stabilize and produce membrane proteins (MPs) suitable for cryo-EM and/or X-ray
diffraction of complexes. Prioritized complexes will be assayed as to homogeneity, purity and stability.
Purification will be optimized for structure determination by cryo-EM imaging, which is particularly well suited to
complexes of relatively large size, and for crystallization. Crystallization will use detergent-lipid screens,
bicelles, nanodiscs, cubic and sponge phase crystallizations (Core 6).

## Key facts

- **NIH application ID:** 9993240
- **Project number:** 5P50AI150476-14
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Robert M Stroud
- **Activity code:** P50 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $136,262
- **Award type:** 5
- **Project period:** 2007-08-27 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9993240

## Citation

> US National Institutes of Health, RePORTER application 9993240, Membrane Protein Expression, Purification (5P50AI150476-14). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9993240. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*