Strategies to Increase Transient Protein Expression 


In both fundamental and practical biomedical research, recombinant proteins are critical tools. Production of recombinant proteins in required numbers and purities for various purposes has grown more common thanks to recombinant DNA (rDNA) technology innovations. Furthermore, scientists may use this technology to design proteins for specific research purposes.  

Because each protein has its own structural and functional features, particular methods must be established to maximize its expression and purification. This application note will cover major parameters impacting transient protein expression output in a mammalian system and critical tactics for modifying them to generate functional proteins in the desired amount and purity. 

Conditions for Protein Expression 

Purity, quantity, and functionality are essential factors in recombinant protein expression. In addition, protein aggregation, insolubility, or misfolding can result from a range of issues, including inadequate sequence design and vector selection, contamination, inappropriate reagents, and experimental conditions, all of which can influence recombinant protein production. 

Because of the transient nature of this process, optimizing it is essential for improving custom protein expression, consistent quality, and repeatability. Several tactics may be employed to improve productivity, and this article will go through some of the more popular ones. 

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Developing an Expression Vector 

To prevent many of the difficulties stated above, create the optimal coding sequence and find the most suitable expression vector for your downstream application. When you start your expression, you can save time, effort, and money by using powerful codon optimization algorithms like OptimumGeneTM and expression vector choosing guidelines like GenScript’s GenSmartTM Design Tool. Regardless of your sequence or expression method, the following measures can help prevent or manage inadequate protein expression difficulties. 

Maximize Protein Solubility 

Poor protein solubility is a significant contributor to low recombinant expression yield. Therefore, we propose that you experiment with some of the typical expression condition settings to improve protein solubility before altering your sequence and expression vector: 


HEK293 and other mammalian cell lines should be grown at 37°C in a 5% CO2 incubator. However, recent research has found that cooling HEK293 cells from 37°C to 33°C for at least the length of a full day (24hrs) after transfection can boost protein expression 1,2.  

However, it is well established that lower temperatures slow cell development; certain researchers have found that lower temperatures increase cellular productivity in the protein manufacturing system. As a result, it can boost recombinant protein production while also reducing the degradation of proteolytically sensitive proteins. 

Growth Medium 

Batch culture is the most frequent method for cultivating cells for recombinant protein production. As a result, all nutrients essential for cell development must be provided by including them in the growth media. Although different suppliers offer identical base media, minor variances might impact the outcome of your specific expression cell line. 

Also, ensure the pH, concentration, type, and even lot number or batch of the external components you add to growth media, such as serum, are optimized. 

Media Supplements 

In some circumstances, adding a particular chemical to the growth medium to boost protein expression is indicated. One example is to decondense chromatin with histone deacetylase inhibitors and encourage integrated gene transcriptional activity. Another instance is the co-transfection of growth factors and cell cycle regulators, like acidic fibroblast growth factors, which have also been found to boost protein production. 

Leveraging a Fusion Partner 

Gene fusion partner technology is another way to improve the expression of low-yield proteins. To boost the solubility of your protein, you can use promoters, enhancers, tags, and other fusion components. Protein tags are short peptide chains mounted onto a recombinant protein and are typically removed after protein production by enzymatic or chemical processes. Several common tags may be used in experiments to boost protein expression.  

Several criteria determine the proper tag for a given expression system. The cost, the binding and regenerating capacity of affinity resin, the condition of the purification buffer, and the final purpose of the experiment are the most important factors to consider. For example, if you want a high yield, you should employ solubility tags like GST, having strong translational initiation signals and may induce numerous expression levels. If you need a lower level of expression, a more strict epitope tag, such as the His tag, is recommended. 

Boosting Purification 

The final and most important strategy is to improve your purity method. Purification of recombinant proteins can be done manually or using a chromatography device. Different purification procedures might be planned out depending on your intended use and purity criteria.  

For example, a 1-step affinity purification approach will deliver enough protein and purity for a range of downstream applications if protein expression is strong. Affinity purification is usually followed by Size Exclusion Chromatography if further purification is necessary (SEC). Also, additional purification can become possible through Ion Exchange (IEX) Chromatography. 


Companies resort to temporary protein synthesis to shorten timelines, obtain immediate access to protein for early go-no-go judgments, delay establishing stable cell lines, and save money. The capacity to produce the requisite amounts of quality proteins — typically ranging from milligram to several grams of protein — in the appropriate timeframes is critical to expanding the role of transient protein synthesis within the biotherapeutic discovery and development workflow. As a result, boosting the efficiency and scalability of processes are essential features of manufacturing. 

To improve transient protein expression, researchers must evaluate the technique of transfection, which impacts both productivity and process scalability.