Recombinant Protein Expression and Purification Analysis
If you plan to use the protein of interest in downstream experiments, it is essential to successfully express the recombinant protein. Optimizing the solubility of the protein at the early stage can significantly reduce purification time and effort later on. 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 protein solubility, simplify purification, and not interfere with downstream applications. While there are many cleavage sites available, your main concern is which tag will enhance solubility, expression, and purification of the target protein. Remember, the tag can always be removed if it might affect the protein’s function. Some tags, like glutathione-S-transferase (GST) or maltose-binding protein (MBP), are known to increase solubility. However, they are relatively large (around 26kDa and 41kDa respectively). Smaller tags such as hexahistidine, FLAG, or streptavidin are often used for easier capture and purification. It's a good idea to test a few different tags and clone them. If possible, use a C-terminal tag so that the purified protein remains full-length without any truncation.Soluble Protein
A common issue when expressing recombinant proteins is that eukaryotic proteins often become insoluble when expressed in bacterial systems. This leads to denatured, aggregated, or truncated forms that end up in inclusion bodies. These require additional purification and refolding steps, which may not be efficient or complete. Here are three key parameters for obtaining soluble, functional 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 for solubility, as they could disrupt proper folding. Try varying the concentration to find the optimal one for soluble expression.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 first grow the culture at 37°C, let it cool down to the induction temperature before adding IPTG.Induction Time
Longer induction times aren't always better, especially for toxic proteins. Sample at regular intervals during induction to monitor expression. Typical induction times vary by temperature: 4 hours at 37°C, 5–6 hours at 30°C, and overnight for temperatures below 25°C.Pre-test
Clone the coding sequence of your protein into 2–3 vectors with different tags (e.g., hexahistidine, GST, MBP). Test various induction times, temperatures, and IPTG concentrations in small-scale pre-experiments. After induction, analyze: (1) uninduced samples, (2) induced samples at different time points, (3) lysates, and (4) soluble fractions.Still Not Getting Soluble Protein?
If the above strategies don’t work, consider these alternatives: - Express only part of the target protein: Smaller domains are often more soluble than the full-length protein. - Use a different expression system: This may help preserve post-translational modifications. - Purify under denaturing conditions: Although not ideal, this method can sometimes be the only solution. Denaturing agents like urea or guanidinium hydrochloride can improve purity but require careful refolding afterward. Many protocols exist for refolding inclusion body proteins.JDC Series - Charging Post Power Module Connector
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