Recombinant proteins are essential tools in basic and applied biomedical research. Using the power of recombinant DNA (rDNA) technology, production of recombinant proteins in desired quantities and purities for a variety of applications has become more widespread. Moreover, this technology enables scientists to engineer proteins for specific study objectives. Since every protein has unique structural and functional characteristics, tailored protocols must be developed to optimize the expression and purification of each individual protein. In this application note, we will describe key factors affecting transient protein expression yield in a mammalian system and discuss essential strategies to modify them in order to obtain functional proteins in desired quantity and purity.
Three aspects are key in recombinant protein expression: quantity, purity, and functionality. A variety of factors such as poor sequence design and choice of vector, contamination, suboptimal reagents and experimental conditions can lead to protein insolubility, aggregation, or misfolding, which ultimately affect recombinant protein expression.
The strategy to avoid many of the above-mentioned problems is to design the best coding sequence and choose the most appropriate expression vector for your specific downstream application.
Low protein solubility is a key factor in obtaining a low yield in recombinant expression. Before redesigning your sequence and expression vector, we recommend you to first manipulate some of the common expression condition parameters in order to enhance protein solubility. Please click download to see more details about the condition parameters.
Another strategy to optimize the expression of low-yield proteins is the gene fusion partner technology. This strategy utilizes promoters, enhances, tags, and other fusion elements to increase the solubility of your protein.
Protein tags are short peptide sequences that are grafted onto a recombinant protein and are usually removed by enzymatic means or by chemical agents at the end of protein expression. There are several common tags that one can use in ones experiment to enhance protein expression. For more information on common tags, click download.
The last essential strategy is on improving your purity method. Recombinant proteins can be purified manually or by using a chromatography system. Depending upon you desired application and purity requirements, different purification methods can be planned out. If protein expression is robust, a 1-step affinity purification method will yield sufficient protein and adequate purity for a variety of downstream applications. If further purification is required, affinity purification can be typically followed by Size Exclusion Chromatography (SEC). For further purification, an Ion Exchange (IEX) Chromatography can be used.
For more information, please click download.
