Recombinant protein expression and purification analysis
If you plan to use the protein of interest in downstream experiments, it's crucial to achieve successful recombinant protein expression. Optimizing the solubility of the protein at an early stage can save a significant amount of time during purification. Here are some recommendations for recombinant protein expression and purification:
What’s in the label?
Choosing the right tag can be challenging. The ideal tag should help improve solubility, simplify purification, and not interfere with downstream applications. While there are many cleavage sites available, your main concern is which tag will aid in solubilization, expression, and purification of the target protein. Remember, tags can usually be removed if they affect the protein’s behavior.
Tags like glutathione-S-transferase (GST) or maltose-binding protein (MBP) are known to enhance solubility but are relatively large (around 26kDa and 41kDa, respectively). Smaller tags such as hexahistidine, FLAG, or streptavidin are often used for capture and purification. Try using different tags and clone them into your system. If possible, use a C-terminal tag to ensure that the purified protein remains full-length without truncation.
Soluble protein
A common issue when expressing eukaryotic proteins in bacterial systems is their tendency to become insoluble. This often leads to the formation of inclusion bodies, which require further purification and refolding—processes that may be inefficient or incomplete.
Here are three key factors to consider for obtaining soluble natural proteins:
IPTG concentration
The lac operon system controls protein expression. Adding isopropyl β-D-1-thiogalactopyranoside (IPTG), a lactose analog, induces expression. It's best to induce during the logarithmic growth phase, when the optical density at 600 nm (OD600) reaches 0.4–0.6. High IPTG concentrations (>1 mM) may not always yield the best results, as they can stress the cell and reduce solubility. Experiment with different concentrations to find the optimal one for soluble protein production.
Temperature
While some proteins can be induced at 37°C, lower temperatures often improve solubility. Try inducing at 30°C, 25°C, or even 18°C. If you grow the culture at 37°C first, allow it to cool to the induction temperature before adding IPTG.
Induction time
Longer induction times aren't always better, especially for toxic proteins. Take samples every few hours to monitor expression. Typical induction times vary: 4 hours at 37°C, 5–6 hours at 30°C, and overnight for temperatures below 25°C.
Pre-test
Clone the target gene into 2–3 different vectors with various tags (e.g., hexahistidine, GST, MBP). Test different induction conditions: time, temperature, and IPTG concentration. Conduct small-scale pre-experiments and analyze: (1) uninduced samples, (2) induced samples at different time points, (3) lysates, and (4) soluble fractions.
Still no success?
If the above steps don’t work, consider these alternatives:
- Express only a part of the target protein—smaller domains are often more soluble than the full-length version.
- Try different expression systems, which may preserve post-translational modifications.
- Purify under denaturing conditions, such as high concentrations of urea or guanidinium chloride. While this method can yield purer samples, refolding may be required. Fortunately, many protocols exist for refolding inclusion body proteins.
By carefully optimizing each step, you can significantly increase your chances of obtaining a functional, soluble recombinant protein suitable for downstream applications.
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