Prokaryotic expression refers to a method that utilizes gene cloning technology to introduce a target gene into a host cell via an expression vector, enabling stable expression within a prokaryotic organism to obtain large quantities of the desired protein. By reviewing relevant literature, this article summarizes common issues encountered during prokaryotic expression experiments and analyzes potential causes along with corresponding solutions.

Question 1: Truncated protein/no protein expression is observed.

Answer: Possible reasons include：1.The protein was hydrolyzed;2.The codon bias of E. coli caused incorrect translation initiation;3.The gene itself contains unusual structural features.

Solutions：1.Use a vector with fusion tags at both ends to separate full-length and truncated proteins during purification;2.Optimize codon usage;3.Use strains with rare tRNAs, such as Rosetta 2, Rosetta-gami 2, Rosetta-gami B, or RosettaBlue.

Question 2: Inclusion bodies appear.

Answer: The formation of disulfide bonds is difficult/gene expression is too fast, and the expression level is too high.

Possible solutions include: 1. Controlling and optimizing expression levels; 2. Lowering the temperature; 3. Optimizing IPTG concentration.

Question 3: The protein is inactive.

Answer: The possible reason is that the protein is misfolded.

The solution is the same as for Question 2.

Question 4: Cell death, extremely difficult growth.

Answer：The possible reason is that the target protein is toxic and affects the host cells.

Potential solutions：1.Stricter control of basal expression;2.Try using BL21(DE3)pLysS strains.

Question 5: How to improve the purification of His-tagged protein with excessive impurity bands?

Possible solutions：1.If purifying from the supernatant: The target protein may be partially degraded by proteases.  Adding a cocktail of protease inhibitors can help mitigate this issue;2.Increase the imidazole concentration in the binding buffer to reduce non-specific binding of contaminating proteins to the nickel column;3.If impurities co-purify with the target protein: Adding a detergent (e.g., 1–2% Triton X-100) before sonication may help disrupt unwanted protein interactions.

Question 6: What should I do if the nickel column turns brown during use?

Answer: This issue primarily occurs due to the presence of DTT (dithiothreitol) in the buffer. DTT can significantly affect the color and purification efficiency of the nickel resin. For this reason, all nickel resin manufacturers strongly recommend minimizing or avoiding the use of DTT (most nickel resins can tolerate<5 mm="" dtt="">

Question 7: What should I do if the nickel column is clogged?

Answer：1.If the column becomes clogged, it may be due to cell debris or other particulate impurities in the sample. Therefore, ensure the sample is centrifuged at high speed;2.If protein denaturation occurs during supernatant purification, leading to the formation of flocculent precipitates, immediately add 1-2 mM DTT (sample processing should be performed on ice). If the issue persists, add urea to denature the protein and maintain it in a denatured state;3.During sample preparation, avoid using too small a solid-to-liquid ratio, as this can increase viscosity or cause protein precipitation/denaturation. A suitable solid-to-liquid ratio should be between 1:10 and 1:15.

Question 8: What should I do if the solution turns turbid during protein purification?

Answer：1.The appearance of turbidity indicates that the protein is in an unstable environment or is inherently unstable. Therefore, it is necessary to check whether the buffer system is correct, monitor the ambient temperature, or add the reducing agent DTT to the buffer;2.Adding sodium sarcosinate rapidly denatures the protein, eliminating the turbidity.

Question 9: What could be the reasons for failing to elute the His-tagged protein?

Answer：1.Incorrect sonication power (too high, causing protein carbonization; too low, failing to release the protein);2.Incorrect sample or binding buffer composition;3.The histidine tag is not fully exposed;4.Loss of the His-tag.

The following approaches can be taken：1. Adjust the sonication power and add lysozyme before sonication;2. Check the pH and composition (e.g., EDTA) of the sample and binding buffer;3. Perform purification under denaturing conditions (using 8M urea, 6M guanidine HCl, or 1% SDS) with the addition of 1-2 mM DTT;4. Verify His-tag expression via Western Blot (WB) or anti-His antibodies;  optimize upstream construct design, modify His-tag position, or increase the number of His residues (typically 6-10) if necessary;5.Extend incubation time, reduce flow rate, and increase binding time;6.Test different chelating metal ions to identify the optimal binding metal ion.

Question 10: What should I do if the protein cannot be eluted from the column?

Answer：1.The elution conditions are too mild;2.If using low-pH elution, note that a pH below 3.5 may cause nickel ion leakage from the resin;3.The protein may have precipitated on the column.

The following methods can be employed：1.Increase the gradient elution of imidazole or lower the pH;2.Change the elution method by using competitive elution with imidazole;3.Reduce the loading amount and incubation time, try using detergents (1%-2% Triton X-100), adjust the NaCl concentration, or perform elution under denaturing conditions (using 8M urea or 6M guanidine hydrochloride). Alternatively, 2mM DTT or 0.5% sodium sarcosinate can be added to the elution buffer;4.Add a nonionic detergent to the elution buffer (e.g., 2% Triton X-100) or increase the NaCl concentration.

Question 11: How can the sample be treated for preliminary purification?

Answer：1.For supernatant sample processing: Add detergents, protease inhibitors, and reducing agents, while also paying attention to temperature and sonication parameters;2.To remove E. coli endogenous proteins (two bands at 30–40 kDa): Resuspend in a non-denaturing buffer (with added detergent) and sonicate, followed by centrifugation at 6,000 rpm for 30 min at 10°C. Repeat if necessary for better results;3.For washing E. coli inclusion bodies: Wash multiple times with inclusion body wash buffer or use stepwise solubilization with 1M/2M/4M/8M urea, then select the fraction with the highest purity;4.Ammonium sulfate precipitation can be used for preliminary purification.

KMD Bioscience possesses a well-established prokaryotic expression and purification system, capable of providing clients with diverse expression vectors and host strains to deliver optimal expression strategies. Additionally, we offer a wide range of purification methods to ensure high-quality purification solutions.Clients only need to provide us with a protein sequence, CDS, or protein name, and we can efficiently complete the expression and purification of the target protein within a short timeframe, offering a comprehensive one-stop service.