In the field of biotechnology, protein production plays an essential role in scientific research. From drug development to enzyme production, they all rely on efficient and stable protein expression systems. However, when specific genes need to be expressed, problems such as low expression levels, insufficient protein activity, and susceptibility to degradation often occur.
One of the factors affecting these is the codon. It directs the addition of amino acids and protein synthesis. Therefore, if there are different codon usage preferences in the host cell or codon errors in the target gene, they can affect protein expression.
This article explores the effects of codons on protein expression and explains the considerations for codon optimization to help enhance the protein expression level!
About Protein Expression
Protein expression refers to the process of synthesizing specific proteins in organisms by expressing target genes.
1. The Importance of Protein Expression
Protein expression technology has multiple applications in fields such as biopharmaceuticals, disease research, agricultural production, etc.
● Biopharmaceuticals: Protein expression is used for producing drugs such as insulin, antibodies, and vaccines. Through genetic engineering technology, the genes encoding these proteins can be inserted into the expression systems to produce proteins for drugs on a large scale.
● Disease Research: By studying the disease-related protein expression, researchers can understand the development process of diseases and discover potential therapeutic targets. In addition, abnormal protein expression may cause specific diseases, and these proteins can serve as biomarkers for diagnosing diseases.
● Agricultural Production: In the field of agriculture, by inserting specific genes into crops, the disease resistance, nutritional value, or yield of crops can be improved. In addition, protein expression technology can also help research biopesticides for crop protection.
2. Common Issues in Protein Expression
However, some problems encountered in the expression process may affect the yield and quality of proteins.
● The expression level of a target protein may be lower than expected due to problems during gene transcription or translation.
● Interactions between proteins may lead to protein aggregation.
● Incorrect polypeptide chain folding leads to protein inactivation.
These problems greatly limit the application of protein expression in biotechnology.
2. How to Improve Protein Expression?
What affects the protein expression level? Codon is one of the important factors. If the target gene contains many codons that are not commonly used by the host cell, it may lead to a slowdown or early termination of translation, thus affecting the level and quality of protein expression.
The Role of Codon Optimization in Protein Expression
What is codon optimization? Codon optimization is a key technique in genetic engineering to enhance the expression efficiency of foreign genes. By adjusting the codons in a gene sequence to match the host cell's preferences, this process can significantly increase protein expression levels, improve mRNA stability, reduce translation errors, and ultimately boost the yield of the target protein.
1. How to Optimize Codons?
Common measures for codon optimize include redesigning genes using preferred codons, simplifying the secondary structure of mRNA, adjusting GC content, eliminating restriction enzyme cleavage sites, optimizing repeat sequences, and adjusting stop codons.
2. The Advantages of Codon Optimization for Protein Expression
Given the importance of protein expression in biotechnology research, codon optimize for target genes is particularly important, as it can:
● Improve Protein Expression Level
Codon optimization helps to enhance the protein expression level of target genes in organisms. In biotechnology research, there is a growing demand for rapid, high-throughput proteins and antibodies. Improving the expression level is directly related to production efficiency and cost control.
● Reduce Incorrect Protein Folding and Aggregation
Incorrect folding or aggregation may lead to protein inactivation or the formation of insoluble aggregates. Codon optimization helps promote proper protein folding, faster synthesis rates, and less waste.
● Improve the R&D Efficiency in the Field of Biomedical Research
In biomedical research, rapid and efficient access to high-quality functional proteins enables researchers to obtain experimental materials faster. Codon optimize not only reduces the risk of experimental failure but also facilitates the development of new drugs and treatments.
Factors to Consider in Codon Optimization
Codon optimization is a complex that requires consideration of several factors to ensure that the gene is effectively expressed in the host cell. It includes:
1. Codon Usage Bias of Host Species
Same amino acid can be encoded by different codons, leading to codon usage bias. The codon adaptation index (CAI) is a measure to analyze the frequency of preferred or optimal codons in the host cell. A CAI value closer to 1 indicates a better match. (Masłowska-Górnicz et al, 2022, p. 194826)
What determines the matching degree is the tRNA quantity and concentration in the host cell. Therefore, designing the gene sequence according to the kind of tRNA in the host cell helps to recognize codons quickly and improve the expression efficiency.
2. mRNA Stability
Complex and stable mRNA secondary structures may hinder ribosome binding, thereby reducing translation efficiency. Moreover, unstable mRNA sequences are susceptible to degradation. Thus, reduced mRNA concentration will affect protein expression efficiency. (Nouaille et al, 2017, pp. 11711–11724) Therefore, using codons preferred by the host cell can accelerate ribosome binding, reducing the risk of forming stable secondary structures.
3. GC Content
For DNA with a high GC content, the three hydrogen bonds between GCs make the DNA double-strand more stable compared to the two hydrogen bonds between ATs. It may affect DNA replication and transcription processes. (Wikipedia, 2024)
In addition, high GC content also causes more stable and complex mRNA secondary structures, hindering ribosome recognition and binding. Therefore, proper analysis of GC content is needed during the codon-optimized process.
4. Restriction Enzyme Sites
During protein expression, there may be some cryptic splice sites on mRNA. Once activated, it can result in mis-spliced of the mRNA. Then, the amino acid sequence can be changed, causing deletion or early termination, which affects the expression level. (Watts, Anshul et al, 2021, p. 100899) Therefore, during the codon-optimized process, the reduction of cryptic splice sites also needs to be considered.
5. Similar Codon Repeats
Similar codon repeats on mRNA may also affect gene expression. Firstly, it may lead to a slower translation process. Secondly, similar codon repeats may increase the expression error rate when ribosomes recognize and bind similar codons, ultimately forming proteins with abnormal functions. Therefore, this problem needs to be avoided for codon optimize.
6. Stop Codon
During the codon-optimized process, pay attention to hidden stop codons to prevent misreading, causing premature termination of translation. Unexpected termination may result in functional loss or even harmful proteins.
Given the necessity, importance, and complexity of codon optimize, it is crucial to choose a gene synthesis service provider that provides advanced codon optimization technology.
Tsingke as the Protein Expression Service Provider
Tsingke is dedicated to providing the best protein expression services in the industry, and we offer free codon optimization. This complimentary service enhances protein yield and quality, ensuring that your project achieves the highest possible success. By optimizing codon usage tailored to your chosen expression system, we help you save time and resources while maximizing the efficiency of your research. With Tsingke's expert support and advanced technology, you can easily achieve breakthrough results in life science research, drug discovery, diagnostics, industrial production, and bioenergy. Trust Tsingke to accelerate your scientific success.
Conclusion
Overall, codon optimization plays an indispensable role in improving protein expression levels, reducing protein folding and aggregation, and improving R&D efficiency in the field of biomedical research. Efficient protein expression facilitates the development of biopharmaceuticals, vaccine development, and other fields that require large amounts of protein production.
Tsingke's recombinant protein expression that provides free codon-optimized service has earned the trust of many customers for our high expression yields, high quality, high throughput, and fast turnaround times. Contact us for more information!
References
[1] Liu, Y., Yang, Q. and Zhao, F. (2021) 'Synonymous but not silent: the CoDon usage code for gene expression and protein folding,' Annual Review of Biochemistry, 90(1), pp. 375–401.
[2] Masłowska-Górnicz, A. et al. (2022) 'A large-scale analysis of codon usage bias in 4868 bacterial genomes shows association of codon adaptation index with GC content, protein functional domains and bacterial phenotypes,' Biochimica Et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1865(6), p. 194826.
[3] Nouaille, S. et al. (2017) 'The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression,' Nucleic Acids Research, 45(20), pp. 11711–11724.
[4] Wikipedia. (2024) GC-Content [Online]. Available at: https://en.wikipedia.org/wiki/GC-content. (Accessed: 30 August 2024)
[5] Watts, Anshul et al. (2021) 'Optimizing protein expression in heterologous system: Strategies and tools,' Meta Gene, 29, p. 100899.
