Please join us for a discussion of methods to diagnose, correct, and prevent some of the problems that arise when expressing recombinant proteins in E. coli. Features in the plasmid vector and host strain that provide control over basal expression, produce high levels of target protein, and improve solubility of recombinant proteins will be identified. The influence of downstream processing on yield will be discussed. We will share some techniques for improving purity using affinity chromatography resins.

A variety of methods for protein expression in insect cells will be discussed. The reliable baculovirus expression system is a standard procedure to produce large quantities of recombinant protein. The BacMagic™ method provides a selection for recombinant virus, eliminating the need to screen for recombinant plaques and saving about one week compared to conventional methods. For even faster results, the Insect Direct™ expression plasmids contain an enhanced AcNPV immediate early promoter for expression directly in Sf9 cells without viral infection. Target proteins are purified from the insect cells only 2 days after transfection with yields comparable to those from a traditional baculovirus system.

The methods used in our lab for rapidly cloning expressing, and purifying a group of kinases and phosphorylation targets will be presented. PCR products are directionally inserted into several expression vectors by ligation independent cloning (LIC). For optimal bacterial expression, different combinations of vectors and hosts are evaluated for each protein. Induction of expression using components in the bacterial growth medium requires no monitoring of cell density or addition of IPTG to the cells, meaning more samples can be tested in parallel. The insect expression plasmids contain an enhanced AcNPV immediate early promoter for expression directly in Sf9 cells without viral infection. Target proteins are purified from the insect cells only 2 days after transfection with yields comparable to those from a traditional baculovirus system.

Methods for enhancing protein solubility using vector-encoded tags and specialized bacterial expression hosts will be discussed. The importance of temperature, medium, and induction conditions are described, as well as co-expression of multiple proteins. One option for proteins that remain insoluble in bacteria is expression in insect cells. Another option for the difficult target proteins expressed as inclusion bodies is a 96-well screen to rapidly determine the buffer conditions necessary for target protein refolding.