Prokaryotic expression refers to the method of using gene cloning technology to convert foreign target genes into host cells by constructing an expression vector to make them stably expressed in prokaryotic organisms, to obtain a large number of target proteins. By reviewing relevant literature, this paper summarizes the common problems in the process of prokaryotic expression experiments and analyzes the possible causes and solutions.

Question 1: Scut-out Protein/no Protein Expression.

Answer: 1. The protein is hydrolyzed; 2. The codon preference of E. coli makes the translation start site wrong; 3. The gene itself has a special structure.

It can be done by: 1. using a vector with a fusion tag at both ends, which can distinguish full-length proteins from truncated proteins during elutions; 2. optimizing codons; 3. using rare codon strains Rosetta 2, Rosetta-gami 2, Rosetta-gami B, RosettaBlue.

Question 2: The Emergence of Inclusion Bodies.

Answer: Difficulty forming disulfide bonds / Gene expression is too fast, the amount of expression is too high.

It can be done by: 1.The expression level can be optimized by control; 2.Cooling; 3.IPTG concentration optimization and other methods to solve.

Question 3: Protein is Inactive.

Answer: The protein may be misfolded.

The solution is the same as problem two.

Question 4: Cell Death and Extremely Difficult to Grow.

Answer: The possible reason is that the protein of interest is a toxic protein that affects the host cell.

It can be done by: 1. Stricter background expression; 2. PlysS strains can be tried.

Question 5: There are Many Protein Hetero-bands Purified by His Tag, How Can I Improve it?

Answer: 1. If the purified supernatant is purified, it may be that the protease partially degrades the protein of interest, which can be improved by adding a variety of protease inhibitors; 2. It can increase the concentration of imidazole at the beginning of heteroprotein binding to the nickel column to reduce the affinity of hetero-protein and nickel column; 3. Heteroprotein and target protein binding, which can be eliminated by adding detergent before ultrasound (1%-2% Tritonx-100).

Question 6: What Should I do If the Nickel Strain Appears Brown During Use?

Answer: This situation occurs, mainly due to the influence of DTT (dithiothreitol) in the buffer, DTT will have a great impact on the color and purification efficiency of the nickel column, so all nickel column manufacturers emphasize avoiding the participation of DTT as much as possible (general nickel column tolerance is less than 5 mM DTT, it is recommended not to exceed 2 mM DTT in the buffer).

Question 7: What Should I Do If the Nickel Strain is Blocked?

Answer: 1. Column blockage may be caused by cell debris or other debris in the sample, so the sample must be centrifuged at high speed; 2. During supernatant purification, the protein is denatured, and flocculent is produced, quickly add 1-2 mM DTT (supernatant sample processing should be carried out in an ice bath), and do not add urea denaturation, so that it is in a denaturing environment; 3. The material-liquid ratio during sample processing should not be too small, otherwise the viscosity is large, or lead to protein precipitation or denaturation, and the material-liquid ratio should be between 1/10-1/15.

Question 8: What Should I Do if There is Turbidity During Protein Purification?

Answer: 1. The presence of turbidity indicates that the protein is in an unstable environment or is unstable itself, so check whether the buffer system is correct, the ambient temperature, or add the reducing agent DTT to the buffer; 2. Add sodium sarcosinate to quickly denature the protein and eliminate turbidity.

Question 9: What is the Reason for the Failure to Elute to the Protein with the His-tag?

Answer:1. The power of the ultrasound is not right (too large, the protein is charred, too small, the protein is not released); 2. The sample or binding buffer is incorrect; 3. The tag of histidine is not fully exposed; 4. His tag is missing.

It can be done by: 1. Change the ultrasound power and add lysozyme before ultrasound; 2. Detection of pH and composition of the sample and binding buffer (EDTA); 3. Purification under denaturing conditions (with 8M urea, 6M guanidine hydrochloride, 1% SDS) and adding 1-2mMDTT; 4. WB or anti-his antibody to check whether His is expressed, build upstream, change the site of his-tag, and increase the number of his if necessary (commonly used 6-10); 5. The incubation time is not enough, reduce the flow rate and increase the incubation time; 6. Change the chelated metal ions to find the best binding metal ions.

Question 10: What Should I do If the Protein does not Elute on the Column?

Answer: 1. The elution conditions are too mild; 2. The method of reducing PH elutes because if the pH is lower than 3.5, it will cause nickel ions to fall off; 3. The protein has been precipitated on the column; 4. Non-specific hydrophobic or other reciprocal reactions.

It can be done by: 1. Increase gradient elution of imidazole or decrease pH; 2. Change the elution approach, imidazole competitive elution; 3. Reduce loading and incubation time, try decontamination (1%-2% Tritonx-100) or change the concentration of NaCl, or elute under denaturing conditions (with 8M urea, or 6M guanidine hydrochloride), and eventually add 2 mM DTT or 0.5% sodium sarcosinate to the elution buffer for elute; 4. Add a nonionic detergent to the elution buffer (e.g., 2% Triton X-100) or increase the concentration of NaCl.

Question 11: How to Deal with the Sample to Make it Initially Purified?

Answer: 1. For supernatant sample processing, detergent, protease inhibitor, and reducing agent should be added, and the temperature and ultrasonic crushing parameters should also be paid attention to; 2. The E. coli self-belt (two 30 Kd-40 Kd) can be suspended ultrasonically with non-denaturing buffer (add detergent), centrifuge at 6000 r/min, 30min, 10 °C. (If the effect is not enough, continue with the above steps); 3. The washing of E. coli inclusion body can be washed several times with inclusion body lotion, or 1M/2M/4M/8M urea can be gradually dissolved, and the best purity can be selected;4. Ammonium sulfate precipitation method can be used for preliminary purification.

KMD Bioscience has a complete prokaryotic expression purification system, which can provide customers with a variety of expression vectors and host strains, can provide customers with a high-quality expression strategy, and we have a wealth of purification methods to ensure a high-quality purification program. Customers only need to provide us with a protein sequence, CDS or protein name, we can complete the expression and purification of the target protein for you in a short time, and provide you with a one-stop service.