Common Problems and Solutions of Prokaryotic Expression of Recombinant Protein-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

Common Problems and Solutions of Prokaryotic Expression of Recombinant Protein

1. What are the specific requirements for the design and construction of prokaryotic expression vectors?

Answer: In the design of prokaryotic expression vectors, it is necessary to select suitable promoters, such as T7, lac, etc., which have high transcriptional activity in prokaryotic cells. Selectively use strong terminators to ensure the precise termination of transcription and avoid read-through. It is also necessary to insert appropriate replication origins into the prokaryotic expression vector to ensure that the vector can replicate stably in the host bacteria.

2. How to improve the expression efficiency of prokaryotic proteins?

Answer: Optimize codons to make them more suitable for the expression system. In the process of constructing prokaryotic expression plasmids, it is necessary to ensure the accuracy and directionality of the sequences inserted into them, as well as the restriction sites at appropriate positions and the design of some protective bases.

3. How can the expression conditions of prokaryotic proteins be optimized?

Answer: The selection of inducers and their concentrations and the selection of induction time, as well as the changes in various parameters such as temperature, pH value, and rotation speed in bacterial culture. At the same time, consider the formulation of the culture medium, such as the ratio of C, N, inorganic salts, etc. and whether additional special nutrients or inducers are needed.

4. How to solve the inclusion bodies in the prokaryotic expression of recombinant proteins?

Answer: Inclusion bodies are formed when prokaryotic proteins are expressed rapidly and folded incompletely. The problem of inclusion bodies can be solved by lowering the induction temperature, adjusting the concentration of the inducer, using GST fusion tags, etc.

5. How to solve the protein degradation in the prokaryotic expression of recombinant proteins?

Answer: The host bacteria's own proteases may cause the target protein to be degraded. You can try to replace the host bacteria and add protease inhibitors to the culture medium.

6. How to solve the low protein expression in the prokaryotic expression of recombinant proteins?

Answer: You can optimize the expression conditions, and you can also re-construct the prokaryotic expression plasmid, replace the expression vector, promoter or perform codon optimization.

7. How to solve the loss of protein activity in the prokaryotic expression of recombinant proteins?

Answer: It may be caused by insufficient post-translational modification or misfolding of the protein. You can try to add auxiliary factors to the culture medium or re-perform in vitro refolding experiments.

8. How to evaluate the expression effect of prokaryotic proteins?

A: SDS-PAGE can show the location of the target protein and preliminarily determine its expression level and purity; Western Blot uses specific binding to further confirm the existence of the target protein; enzyme activity assays or functional experiments verify the biological activity of the protein; mass spectrometry is used to identify the molecular weight, amino acid position, etc. of the protein.

9. How to maintain the stability of recombinant protein?

A: This can be achieved by adding stabilizers, adjusting the pH value and temperature of the environment, and reducing the number of repeated freeze-thaw cycles of the protein. Choose a suitable container and sealing method.

The prokaryotic expression platform optimized by TekBiotech for many years is used to ensure the high level expression of the customer's target protein. At the same time, TekBiotech has a professional technical team that can provide customers with comprehensive technical support and solve all the technical problems mentioned above.

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