Preparing for Characterization: Expression and Purification

When proposing a research project, there’s a number of project steps that require advanced planning. Asking questions like, “do I need to express and purify any protein(s)?”, and accounting for this in your project proposal saves you time and unexpected capital expenditures. In this article, we’ll compare the pros and cons of vital project parameters involved in preparing for downstream characterization so you can hit your research milestones and publish successfully.

Protein Expression

Many projects involve protein production, which can be classified into two major substeps, expression and purification. Choosing an adequate expression system can be tricky and may impact the quality of your end product, protein. There are a number of available systems (including insect, fungal, algal etc.), here we’ve summarized a couple of the most common ones below.

Cell-based expression systems are the most common expression systems and can be performed with either bacterial or eukaryotic cell systems. A simple example of a bacterial cell expression system is E. coli, which is the most well-documented method to express proteins and is an excellent choice for cytosolic and/or excreted proteins. E. coli cultures are quick to grow, easy to keep healthy, can withstand heat shock transfection and are very low-cost, making this expression system quite attractive for most project directions. It should be noted that transmembrane proteins, for example, should not be expressed in E. coli systems; since E. coli does not possess an endoplasmic reticulum, proper structure translation cannot be achieved in many cases. It should also be noted that P. fluorescens can produce high yields of soluble proteins at a low cost.

In contrast however, eukaryotic cell expression (ie. Chinese Hamster ovary cells, CHO) can mitigate these shortcomings observed with bacterial cell systems. Eukaryotes contain an endoplasmic reticulum and will ensure the native conformation of proteins are upheld, including transmembrane or membrane-bound proteins. This makes a eukaryotic expression system a lot more attractive to projects that involve transmembrane protein expression and purification. However, eukaryotic cell expression systems tend to be more sensitive to fatal conditions, do not yield as much product and can cost more money.

Overall, here are some important factors to keep in mind when selecting an appropriate expression system for your project:

  • Do I want to look at protein interactions later in my project?
  • Are my proteins soluble or cytosolic?
  • Do my proteins require any protein tags for downstream assays?
  • Do my proteins require post-translational modifications?
  • How much protein do I need?
  • Is this protein a complex?
  • Is the cost for expression a limiting factor?

With these factors in mind, the expression system you use should be selected on a fit-for-purpose basis.

Protein Purification

Similar to expression, there are a number of methods and factors to consider when selecting an appropriate purification system. We cover some of the main methods below but please note that there are multiple preliminary steps we’ve excluded (ie. precipitation, ultracentrifugation etc.) and one or more purification methods should always be used in conjunction.

Size exclusion chromatography (SEC) will allow for separation of large macromolecules from smaller ones and is an excellent baby step to any chromatographic purification process. Post-centrifugation, very large macromolecules or organelles (ie. whole cells, nuclei) will have been pelleted while smaller macromolecules (ie. soluble proteins) and will reside in the supernatant. Inside the SEC column, chromatographic beads have micropores which allow smaller molecules to migrate inside the pores, making their elution pathway significantly longer than that of larger molecules, which cannot enter the pores. In this respect, the first few chromatographic fractions will contain large molecules while later fractions will contain smaller molecules. Provided the molecular weight of your protein(s) of interest is known, specific fractions can be taken for downstream purification techniques. It should be noted that SEC is a low-tech, low-cost method that will help purify your proteins but likely not to the extent that is often demanded for downstream assays.

Moving forward, affinity chromatography will specifically capture your protein of interest, even out of a fairly complex matrix. When employing affinity chromatography, your protein of interest may have a covalently linked protein tag (ie. biotin, His-tag) and the chromatographic beads may have a highly specific capturing molecule (ie. streptavidin, NTA, respectively). Moreover, a highly specific antibody recognizing your protein of interest may also be conjugated to the chromatographic beads for specific capturing (ie. anti-BSA antibody). When a sample of partially-purified protein (ie. useful fractions collected from SEC) is run through the column, the conjugated beads will capture your protein of interest and allow irrelevant proteins to continue flowing through as waste eluent. Subsequently, an elution buffer or competing compound can be run through the column, which will allow your protein of interest to be eluted and taken as a fraction. Affinity chromatography is a highly specific, time-efficient method to maximize sample purity and protein concentration, but comes at a higher cost. It is also important to note that a hallmark of this purification system requires adequate planning and selection of your expression system. Strategizing employment of protein tags is not only important for techniques like affinity chromatography but also other in vitro assays, such as surface plasmon resonance.

Here are some important factors to keep in mind when developing a purification scheme:

  • Do I know the molecular weight of my protein(s) of interest?
  • How pure does my protein need to be?
  • How much concentration of protein do I need?
  • What tags did I express my protein with?
  • Can I take advantage of these tags during purification?
  • What downstream in vitro assays do I need to carry out?

In our next blog post, we will discuss the variety of techniques employed for identifying potential protein binding partners and show how you can use benchtop surface plasmon resonance to get the binding kinetics data needed to publish. Notably, the OpenSPR is a user-friendly and affordable instrument that provides label-free binding kinetics data at your own lab bench, without the need for a specialized technician.

Find out how benchtop SPR can help you publish faster:

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