Thoughtful construct design is often the difference between a protein that expresses well and one that doesn't. We consult on domain boundaries, tag selection and placement, fusion partners for solubility (MBP, SUMO, Trx), codon optimization, and vector choice for your target expression system. The core maintains a few expression vectors and can guide subcloning, site-directed mutagenesis, and generation of truncation constructs. For challenging targets, we routinely design parallel construct panels to identify the best-behaving variant before scaling up.

E. coli

E. coli remains the fastest and most cost-effective system for producing soluble bacterial proteins and many eukaryotic domains. We support standard and specialty strains to match the demands of your target, including BL21(DE3) derivatives, Rosetta, SHuffle, and C41/C43 strains. Expression is performed in shaking incubators routinely on a 1-6 L scale, with larger scales available if needed. We can produce custom-labeled proteins for downstream experiments on request.

Cell-free expression

Cell-free expression bypasses the need for living cells and is particularly useful for toxic proteins, rapid construct screening, and incorporation of unnatural amino acids. Reactions can be assembled in hours rather than days, making cell-free a powerful option for parallel evaluation of construct variants before committing to a large-scale prep. We can advise on when cell-free is the right choice and coordinate reactions using commercial kits.

We support the full purification workflow from cell lysis through purified and characterized final product. Standard capabilities include affinity chromatography across common tag systems (His, GST, MBP, Strep), ion exchange, and size-exclusion chromatography on our Bio-Rad FPLCs at 4 °C. We can monitor up to 4 wavelengths at once, which is helpful for proteins bound to DNA/RNA or ligands. Tag removal is available using a variety of proteases. Cell lysis is performed by sonication or microfluidization depending on scale and sample sensitivity. Every purification is accompanied by SDS-PAGE and yield reporting — additional QC is available through our biophysical suite to confirm monodispersity and stability before handoff.

Size-Exclusion Chromatography (SEC) separates macromolecules in solution by size and shape. The macromolecular separation profile is determined by various bead pore sizes for the specific column. Large particles will pass through the column more quickly and elute before smaller molecules, which diffuse into the pores and therefore elute later in the run. For globular proteins, SEC can be used to estimate the mass and oligomeric state of the sample by comparison to a set of MW standards. We have two Bio-Rad FPLCs with multi-wavelength detection at 4 C for runs.

Columns: 

• Superdex 75 (24 and 120 mL)
• Superdex 200 (24 and 120 mL)
• Superose 6 (24 and 120 mL)

Instrument: Glacios (Thermo Scientific)

The Glacios (200 kV) is configured with a Falcon 4i camera and Selectris energy filter, fringe-free imaging, Smart EPU with Quality Monitor, and EPU Multigrid. Sample preparation is supported by a Vitrobot Mark IV for rapid, high-quality grid preparation. 

Researchers should contact the Facility Director to discuss project suitability and access options.

Instrument: HT7800 (Hitachi)

We routinely perform and train users in negative stain EM using the microscope available in the Central Microscopy Research Facility (CMRF) at Iowa. Negative stain EM is a rapid technique to assess particle quality and homogeniety prior to cryo-EM.

The University of Iowa belongs to the Molecular Biology Consortium which operates beamline 4.2.2 at the Advanced Light Source (ALS) synchrotron within Lawrence Berkeley National Lab. Considerable beamtime is available through our consortium, so all of our crystal screening and data collection is performed remotely at ALS. Contact the Facility Director for questions about access.

ALS beam status

ALS operating schedule

Small-angle x-ray scattering (SAXS) experiments are performed on the BioCAT beamline at the Advanced Photon Source (APS) synchrotron at Argonne National Lab. We routinely perform SEC-SAXS and SEC-MALS-SAXS, but all available configurations can be found here. User proposals must be submitted to obtain beamtime.

Instrument: Octet RED96 (Sartorius)

Biolayer interferometry (BLI) is a label-free technology used to study biomolecular interactions in real-time. It operates on the principle of interferometry, where changes in the optical thickness of a biomolecular layer at the sensor surface result in interference patterns. BLI involves the immobilization of one binding partner (ligand) onto a biosensor tip. As the tip interacts with the other binding partner (analyte), changes in interference patterns occur, allowing for the determination of binding kinetics, affinity, and specificity. This technique offers advantages such as rapid data acquisition, minimal sample consumption, and versatility in studying various biomolecular interactions.

Instrument: CFX96 qPCR (Bio-Rad)

Differential scanning fluorimetry (DSF) or Thermal Shift Assay uses a fluorescent dye to measure protein conformational changes and unfolding by tracking changes in fluorescence as a function of temperature. The fluorescent dye binds to hydrophobic regions on the protein as it begins to unfold with increasing temperature. Many different dyes can be used, but the most common dye to start with is SYPRO Orange. DSF is commonly used to study protein stability across many buffer conditions and to discover small molecule ligands in drug discovery. Our facility stocks the DSF screens listed below:

• Solubility and Stability Screen (Hampton)
• Solubility and Stability 2 Screen (Hampton)
• RUBIC Buffer Screen (Molecular Dimensions)
• Durham pH Screen (Molecular Dimensions)
• Durham Salt Screen (Molecular Dimensions)

Instrument: DynaPro NanoStar (Wyatt)

Dynamic light scattering (DLS) is a useful technique to measure the size distribution of biomolecules in solution. Sample homogeneity can be rapidly assessed and the hydrodynamic radius determined. DLS measurements are size and shape dependent, so molecular weight estimates may not be accurate. More accurate measurements of molecular weight can be made from static light scattering (SLS). Light scattering is routinely used to assess both protein aggregation and stability.

Instrument: TwoMP (Refeyn)

Mass photometry (MP) is a single-molecule technique that enables the direct measurement of mass and size distributions of unlabeled biomolecules in solution. It works by exposing molecules to a beam of light, and as the single molecules make contact with a glass surface they scatter some of the light. Light that isn't scattered is reflected by the surface. The interference between the light scattered by a molecule and the light reflected by the measurement surface is measured and is directly proportional to the molecule’s mass. The technique requires minimal sample and can quickly assess sample heterogeneity and quality.

Instrument: C-Trap (Lumicks)

The C-Trap single-molecule microscope combines optical tweezers, fluorescence, label-free microscopy, and microfluidics in a nicely integrated solution. Optical tweezers use a highly focused beam of light to trap beads, which can be coated with various biomolecules. Tiny forces can be applied to these molecules and then measured and/or visualized to understand the impact on interactions. Find more info and applications here.

We can predict structures of proteins, protein complexes, protein–nucleic acid assemblies, and protein–ligand complexes from sequence alone. This is useful for generating starting models for experimental work, interpreting mutations, and guiding construct design when no experimental structure is available. We also help interpret confidence metrics (pLDDT, PAE, ipTM) in the context of your biological question.

We can assist or perform de novo design of proteins, binders, and scaffolds using recent generative models and design pipelines. Applications include designing miniprotein binders against a target of interest, engineering novel enzymes, stabilizing therapeutic candidates, and generating custom scaffolds for structural or functional studies. We support the full design → filter → order → test workflow.

Prediction of binding poses and rank compounds against a target of interest. Useful for hit identification, lead optimization, and interpreting results from small-molecule screens.