Recombinant Expression in E. coli
E. coli is perhaps the most widely used host system for recombinant protein expression. The expression level in E. coli is in general much higher than that in mammalian cells. Hence, it’s a cost-effective option for scale-up production. The turnaround time is also much shorter. Most importantly, E. coli can be used to produce proteins that are not easily secreted in the mammalian cells.
Over the years, Bon Opus has developed a collection of proprietary reagents and innovative approaches. Our support team will help you to explore different options and work closely with you each step of the way.
Codon optimization is an important step to increase expression level in all expression system. Bon Opus will help optimize your sequence specifically for E. coli expression.
Choosing Strains and Expression Conditions
There are different strains of E. coli with a variety of productivity and doubling times. Bon Opus carries a large collection of E. coli strains, allowing us to screen for the most efficient expression host for complex projects.
Choosing Expression Vectors
Vectors (and promoters) can also have an impact on expression level. Different vectors can be used to drive expression under different culture conditions. Commonly used vectors include pSumo, pBAD, pET system, pQE system, pGEX system, and pBV220.
To achieve optimized expression, culture media (CD/LB/XY1), and culture temperature (37/30/16) need to be carefully selected. The use of fermentors - over traditional shake flasks - can also improve the outcome. Using Figure 1 to the right as an example, the yield of the E. coli expression can be increased from 0.1mg/ml to 36mg/ml through optimization of culture conditions.
Use of Tags to Promote Secreted Expression
60% of insoluble cases can be solved by introduction of fusion tags. They function by not only facilitating expression, but improving solubility as well. The table on the right summarizes the performance of the common fusion tags based on our prior experience. Commonly used tags include SUMO (facilitated expression by 80% and improved solubility by 70%), GST (facilitated expression by 75% and improved solubility by 65%), and TRX (facilitated expression by 90% and improved solubility by 60%).
In Vitro Refolding
Our team of researchers has many years of experience with refolding. Out of 210 inclusion body cases, the Bon Opus team was able to solve 140 cases by tuning refolding conditions. Figure 2 shows the successful recovery of inclusion body proteins (Left panel, In) using our in-house developed buffer and approaches. The product’s bioactivity has been verified.
This may include the co-expression of chaperone proteins, in addition to periplasmic expression.