Understanding protein production from start to finish

In protein research, going from idea to pure characterized protein takes multiple steps and techniques. When streamlined, this process can help you get results fast, meet publishing deadlines, and make critical decisions on a project.

Most scientists working with exploring proteins and developing specific target biomolecules use several steps including cell culture, sample preparation, purification, and analysis. This requires understanding the principles behind many different tools – and learning these steps takes time that scientists could spend generating data and progressing research. If you’re short on time, setting up a strategy for a complete protein development and production workflow can seem overwhelming.

Scientists at Cytiva Jon Lundqvist, Jinyu Zou, and Anna Moberg have more than two decades of experience in protein research. This article covers their expert guidance, insight, and tips to help you make fast progress in protein research.

From A to Z in the protein production and purification workflow

Over the years, advancing technology has continued to simplify and improve usability across steps in the protein research workflow, enabling individual scientists to cover all or larger parts of it. That’s mostly good news – but it can also mean scientists have less time to learn procedures like cell culture, protein purification, and protein characterization.

To help guide your protein production, Jon, Jinyu, and Anna are combining their expertise to provide insights that can quickly get you up to speed and ready for your next publication or presentation.

Steps in protein research: cell culture, sample prep, purification, and analysis

Before setting up the protocol, set your goals and objectives. To do this, ask yourself questions like:

  • What is the purpose of your research? How will you use the purified protein (e.g., to determine structure and function, for therapeutic or diagnostic use, etc.)?
  • How much information do you have on the identity and function of the target protein?
  • What analysis methods will you use?
  • Do you need post-translational modifications (PTM), such as glycosylation, for protein functionality? Will that influence the expression system you choose?

Clear goals help you decide which expression system to use, the purity and yield to aim for, and the amount and quality of sample you need to conduct multiple analyses. Setting goals will also help you choose which analysis methods to use.

When it comes to choosing equipment and consumables, do some extra research. Different proteins have different properties – so reusing protocols and existing equipment might not provide the outcomes you want. Search the literature in your field, and ask around for more insight. Your colleagues can have a wealth of insight depending on the size of your lab. Conferences, meetings, and webinars can be great opportunities to learn from other scientists. Vendors can also provide methods that are easy to replicate or adapt to your application.

Steps in protein research: cell culture, sample prep, purification, and analysis.

Sample prep

Throughout sample preparation, protein purification, and analysis, you need to avoid protein damage and ensure that proteins maintain activity.

Some proteins are more sensitive than others. Understanding your protein will help you determine which conditions to use and which to avoid. Some proteins, like secreted proteins, have lower expression levels – when expression levels are low, it becomes even more important to make sure you’re maintaining high yield in each step.

Sample preparation depends on protein type. Membrane proteins, protein complexes, and secreted proteins will have different sample preparation protocols. For intracellular proteins, scientists lyse the cells to release the target – you can do this through sonication, homogenization, freezing and thawing, or chemical agents.

For extracellular proteins obtained with mammalian or insect cells, you can directly apply cell harvest. Scientists often perform cell harvest using centrifugation. In most applications, scientists transfer the supernatant to protein purification containing the target protein, or analyze it directly to perform identification and determine activity.

If needed, you can use filtration to remove particles after centrifugation, or before chromatography to protect your columns. Desalting at laboratory scale is a well-proven, simple, and fast method that removes low molecular weight impurities and transfers the sample into required buffer in a single step.

You can use desalting or buffer exchange prior to a chromatography step or between purification steps for sample conditioning.

Protein purification

In protein research, scientists often use two purification stepsaffinity chromatography and size exclusion chromatography. If you need high purity, add an additional intermediate step of ion exchange or hydrophobic interaction chromatography. However, try to use as few steps as possible — adding steps decreases overall protein yield.

Combine chromatography techniques to optimize your protein purification protocol.

Tagged recombinant proteins are usually straightforward to purify. Use an affinity resin that’s suitable for the tag system you’re using, and use the protein’s natural conditions to avoid precipitation and degradation.

You might need to remove the tag after purification. After tag cleavage, you need to separate tag-free target protein from the tag and tag-containing target protein. You can also use on-column cleavage – add protease to the column with the target protein bound to enable one-step cleavage and removal.

Scientists often use affinity chromatography for antibody purification. Affinity chromatography uses the high affinity and specificity of protein A and protein G for the Fc-region of IgG from many species. Protein L, which binds to kappa light chains, is another ligand you can use to purify antibody fragments, IgG, and other antibodies from a wide range of eukaryotic species. For higher purity, scientists often use SEC as a second step.

You’ll need more effort when developing the protocols for nontagged proteins. Experiment planning helps you decide which conditions to test, as well as what to optimize to achieve your desired purity and yield.

Scientists commonly use gravity columns in affinity steps and occasionally use peristatic pumps in other chromatography steps. A protein purification system, often in combination with a prepacked column, delivers significantly more control, enabling you to obtain more detailed and consistent information on your target protein and any impurities. It also provides better column protection.

Protein analysis – identification and characterization

Proteins are diverse in size, structure, and biochemical properties. Scientists use these differences to characterize isolated proteins. You can use multiple techniques to confirm identity and binding activity during protein expression, production, in between steps, and within the purified protein.

A lot of techniques and methods are common in analysis and characterization. UV, SDS-PAGE, and SEC are popular choices for identifying which fractions contain the target protein. Assays for specific proteins like enzymatic assays are available and require a substrate specific to the target protein.

After purification, you can determine structures, binding activities, stability, and more. That’s often done using mass spectrometry in combination with high-performance liquid chromatography (HPLC) columns, amino acid sequencing, X-ray crystallography, cryo electron microscopy (cryo-EM), surface plasmon resonance (SPR), Western blotting, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy.

Western blotting

Western blotting is one of the most established and popular techniques for quantifying and identifying proteins. The method builds an antibody:protein complex – scientists bind the antibodies to membrane-immobilized proteins, and detect the bound antibody with a detection method like chemiluminescence or fluorescence. A sample can be a complex protein mixture like a cell or tissue extract, but it can also be a sample of purified proteins, like a fraction from a purification run.

The Western blot workflow has several steps before analysis:

The Western blot workflow has several steps before analysis.

Scientists apply gel electrophoresis to the sample for protein separation before immobilizing the proteins on a membrane, (e.g., Nitrocellulose or PVDF) following electrotransfer from the gel.

Scientists then incubate the membrane with a primary antibody that specifically binds to the protein of interest, wash to remove unbound antibodies, and conjugate a secondary antibody to an enzyme, a fluorophore, or an isotope for detection. The detected signal from the protein:antibody:antibody complex is proportional to the amount of protein on the membrane.

Traditional total protein staining methods often limit the dynamic range, typically by one or two orders of magnitude for silver and Coomassie staining. In contrast, fluorescence and chemiluminescence detection has a much broader detection window and is quick, easy to use, and highly sensitive.

You can use a CCD-based imager to visualize the blot in both chemiluminescence and fluorescence ranges with high sensitivity and resolution.

Determining mechanism of action using SPR

Understanding the nature of molecular interactions is critical in all areas of the life sciences. Label-free interaction analysis generates data-rich content that can help enhance our understanding of interactions between almost any type of biologically relevant molecules.

  • Do the potential interactants bind to each other?
  • How specific is the interaction?
  • How strong is the binding (i.e., affinity)?
  • How fast is the binding?
  • What are the effects of temperature?
  • How much interactant is there in the sample?
Biacore systems enable real-time analysis of events that are driven and regulated through dynamic interactions between key molecules.

Biological processes are real-time events that are driven and regulated through dynamic interactions between key molecules. End-point techniques like ELISA can only offer a snapshot, and provide basic information like overall binding strength. Affinity depends on the ratio of on- and off-rates, so equal affinity interactions can have very different kinetic properties. Biacore systems enable real-time analysis that can provide the data needed to discriminate these crucial differences.

Sensorgrams from Biacore SPR instruments showing three molecules with identical affinities, but kinetic profiles differing by several orders of magnitude.

Sensorgrams showing three molecules with identical affinities, but kinetic profiles differing by several orders of magnitude. These differences are not visible with end-point analyses.

Biacore sensor chips support analysis of a wide range of interactions. You can use kits and ready-made consumables to save time while ensuring consistent capture of molecules e.g. antibodies and common tags.

We recommend using predefined Biacore run methods with application-relevant settings. Combine them with evaluation software for a good data overview that shortens time to results.

Guidance and tips

You might want a one-size-fits-all approach to your protocols, but this can cost you time in the long run. You need to adjust protocols to fit specific proteins, and add, change, or remove steps for specific targets. One-protocol-fits-all thinking can lead to low expression, poor yield, and time-consuming troubleshooting.

We’re often eager to get started and don’t spend enough time reading the literature or planning. While fast at first, suboptimal protocols will eventually delay our results. Defined goals, clear objectives, and thorough planning will help you get better results faster.

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