In research, the protein production, protein purification, and protein analysis workflows are essential to get from idea to pure characterized protein. Achieving results quickly is important to meet pressures to publish as fast as possible.

In the past, many of the steps and techniques used in protein production were divided in different areas of expertise spread around several people. Over the past decades or so, there has been a shift towards individuals covering the complete workflow involved in protein production (the biotechnological process of producing a specific target protein) rather than just parts of it resulting in less time to learn the principles. Setting up a strategy for a complete protein production workflow may therefore seem overwhelming.

Jon Lundqvist and Jinyu Zou, scientists at GE have spent more than two decades in protein research and here they provide insights and tips on how to make progress in research.

From A to Z in the protein production and purification workflow

Over the years, developments have led to simplification and improved usability for different steps in the protein research workflow, which enables individual scientists to cover all or larger parts of it. This is good news; but, it also allows for less time for training to achieve expertise for the different steps such as gene cloning or protein purification. One person is usually not an expert in all areas, yet many of the procedures used are advanced with numerous conditions influencing the outcomes. There is therefore greater demand today for quick design of protein purification and analysis protocols and assays, ability to learn equipment fast, and easy access of instructions.

5 steps in protein research: gene cloning, cell culture, sample prep, purification, and analysis

Before setting up the protocol, set goals and objectives. Ask yourself these questions:

  • What is the purpose of your research; will the purified protein be used to determine structure or maybe for trial therapeutic use?
  • How will the identity and activity of the target be confirmed?
  • What analysis methods will be used?
  • Are post-translational modifications (PTM), such as glycosylation, needed for protein functionality?

Clear goals will help you to determine what expression system to use and what purity and yield to aim for, so that you get enough sample to conduct multiple analysis.

In protein research various techniques can be applied and it may be difficult to choose from the endless selection of equipment and consumables. Different proteins have different properties and therefore reusing protocols and existing equipment may not provide the desired outcomes. Conduct literature searches within the relevant research area. Your colleagues are often a wealth of information depending on the size of the lab that you are working in. Conferences and meetings are also great opportunities to learn from fellow scientists.

Workflow

Isolation and gene cloning

Once you have found an interesting target (type of protein) for your research, it is time for isolation and genetic cloning of the protein to produce larger quantities for further study. Proteins are expressed in a protein expression system consisting of a host cell (mammalian, bacterial, yeast etc.) and a vector (e.g., plasmid vector) and the recombinant protein may be expressed with or without a tag. The tag is used to simplify identification and purification of the expressed protein investigated. The histidine (his-tag) is most commonly used as this tag is small and can in many cases remain on the protein after purification. While the his-tag is a good place to start, one or more alternative tags must sometimes be tested. The tag selected needs to be soluble and attached to a suitable location on the target protein to be accessible for detection and or binding to suitable purification resin. A cleavage site needs to be engineered into the tagged protein vector if the tag is to be removed post-purification.

A suitable gene expression system needs to be found and matched to the vector before starting to clone the protein. Preferably, cloning is started by screening in 24- to 48-well plates where 1 to 2 mL cultivations are managed in parallel. During the screening, conditions such as temperature, additives, cell culture media etc. are varied. If the expression levels are lower than expected or if the target protein is not expressed you need to go back and look at the tag system, cultivation conditions, etc. and perform further optimization until the clone is found and expression levels are according to set objectives.

Cell culture

Once the expression system and the conditions to grow the host cell are set, scaling up to larger cell culture volumes takes place. For research applications, cell culture volumes of 0.5 to 5 L are commonly used. Cell culture is commonly performed in shaker flasks when working with E. coli if expression levels are high enough. For more control, higher expression levels, and when working with mammalian cells, a fermentor offers better control of conditions such as temperature, glucose levels and pH.

Sample prep

All through sample preparation, protein purification, and protein analysis you need to ensure that proteins are not lost or damaged and activity maintained. Some proteins are more sensitive than others, and you need to know the preferred conditions and conditions that should be avoided. Some proteins, like secreted proteins, have lower expression levels; and when expression levels are low it is even more important to ensure that yield in each sample preparation and purification step is kept high.

Sample preparation will be very dependent on the type of protein. Membrane proteins, protein complexes, and secreted proteins will have different sample preparation protocols. For intracellular proteins, the cells are lysed to release the target; for this, sonication, homogenization, freezing/thawing, or a chemical agent can be used. For extracellular proteins, obtained when working with mammalian or insect cells, cell harvest can be directly applied. Cell harvest is commonly performed by centrifugation. In most applications, the supernatant is transferred to protein purification containing the target protein or directly analyzed for identification and protein activity determination.

Protein purification

Tagged recombinant proteins are usually straightforward to purify. Use an affinity resin that corresponds to the tag system used and the natural conditions for the protein to avoid precipitation and degradation.

Often two stepsaffinity chromatography and size exclusion chromatography—are used. If the requirement for purity is high, add an additional intermediate step of ion exchange or hydrophobic interaction chromatography. However, try to use as few steps as possible as adding steps will decrease overall protein yield.

For non-tagged proteins, more effort is required to develop the protocols. Experimental planning is important to make conscious decisions for what conditions to test, what to optimize, and the requirements for purity and yield.

Gravity columns are common to use in affinity steps and sometimes peristatic pumps in the other chromatography steps, however a protein purification system will deliver significantly more control, obtain more detailed information on target protein and impurities, and gives much better protection of columns.

After purification the tag is removed, although his-tags are often kept as mentioned earlier as they rarely interfere with purification. After tag cleavage, tag-free target protein needs to be separated from the tag and target protein still containing the tag. On-column cleavage can also be used where protease is added to the column with the target protein bound enabling cleavage and removal to be performed in one step.

Protein analysis

Proteins are diverse in terms of size, structure, and properties and the differences can be exploited when separating and identifying proteins. It is instrumental to understand the interactions between molecules, including why and where they occur and the dynamics (kinetics), and the structure of the molecules.

During the protein purification, UV and SDS-PAGE are the most common methods to use for identifying fractions containing the target protein. There may also be assays for specific proteins.

During identification and characterization, separation of proteins by size is commonly performed using electrophoresis and by charge with isoelectric focusing. Mass spectrometry in combination with fractionators such as gas (GC) and high-performance liquid chromatography (HPLC) are used in identification and characterization as well as amino acid sequencing, X-ray crystallography, surface plasmon resonance (SPR), Western blotting, circular dichroism, nuclear magnetic resonance (NMR) spectroscopy, and cryo electron microscopy (cryo EM). In many cases, multiple analysis techniques are used. When working with cells different types of cell analysis are used.

Guidance and tips

You probably want to approach your protocols generically with a one protocol fits all approach, but that is often not the case. Protocols need to be adjusted to fit specific proteins. Steps needs to be added, changed or removed to be suitable for a specific target. One-protocol-fits-all thinking can lead to low expression level, poor yield, proteins that do not elute in the expected fractions resulting in-time consuming troubleshooting.

Many of us are eager to get started and do not spend enough time on literature studies and planning. When there is not enough knowledge there is a risk for suboptimal protocols and delays in results. This is understandable when exposed to pressures to publish. Defining clear goals and objectives and thorough planning will enable you to get quicker results. For example, during protein purification, if you know the purity requirement for your study, you will probably not need to add extra purification steps that make the process longer and potentially reduce protein yield.

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