Pin1 is an enzyme which regulates various proteins necessary for cell cycle progression. It is seen to play an essential role in the cell cycle. However, studies have shown that the upregulation of Pin1 can cause cancer and the downregulation of Pin1 can cause Alzheimer’s disease. Even though the overexpression of Pin1 has cancer-causing abilities, Pin1 has also been shown to have a potential anti-cancer role, through its interaction with p53, a tumor suppressor protein. DNA damage causes phosphorylation and activation of p53, facilitating it’s interactions with Pin1. This interaction leads to the apoptosis of cancer cells and tumor suppression.
In this experiment, the pET expression system was used for cloning and expression of our recombinant protein. The lac operon is present in the pET expression system, allowing the regulation of the expression of the gene of interest. This expression system is commonly used as it is driven by a strong promoter, the T7 promoter, allowing the production of a large amount of protein.
The Pin1 cDNA sequence, with histidine tag, used in this experiment has been cloned into the pET28b+ expression vector and transformed into the Escherichia coli BL21 strain. In this experiment, the transformed E.coli BL21 strain were grown in a bioreactor under controlled conditions to produce recombinant Pin1. At the end of the experiment, the culture was harvested for product recovery of the intracellular protein.
As the recombinant protein is inside the bacteria cell, cell lysis is needed to extract the intracellular protein. There are various cell lysis methods such as sonication and freeze-thaw, which cannot be scaled up and are more effective for small volumes. There are also cell lysis methods such as bead milling and high pressure homogenisation which are suitable for large scale lysis. These methods are very efficient in lysing cells. However, the heat produced during these processes can degrade the proteins.
In this experiment, sonication was used to lyse the bacteria cells. Hearing protection is required during this process as the high frequency noise can harm your ears. Sonication is a type of physical disruption method which uses sound waves with extremely high frequencies (20 kHz) to break open the bacteria cells. Heat created in this process can denature Pin1. Hence, the sample is kept on ice and sonication is done in multiple short bursts.
After sonication and centrifugation, the cell lysate obtained undergoes ultrafiltration to reduce the volume that needs to be fed to the next chromatography process by concentrating the solution.
In this experiment, protein expression was also analysed before and after IPTG induction by quantifying the total amount of all proteins present and the amount of target protein present using spectrophotometry and biolayer interferometry (BLI) respectively.
The BLI-based BLItz system allows the quantification of proteins in just a few seconds, using only a small amount (µL) of sample. The BLItz system makes use of ForteBio’s Dip and Read™ disposable biosensors which is coated with a matrix that is uniform, non-denaturing and minimizes non-specific binding. Interactions on the surface of the biosensor are detected real-time and real-time binding curves are produced. Protein concentration can be evaluated as the concentration of target proteins in the sample is directly proportional to the rate of protein binding to the surface of the biosensor. The concentration of unknown proteins can be determined by comparison to the standard curve. Various disposable biosensors such as Protein A, Protein G, Protein L, Anti-GST, Anti-Human Fab-CH1 and Anti-Human IgG Fc Capture can be used with the BLItz system
Through this experiment, we will reach our objectives to set-up and operate a bacterial fermenter, to collect and evaluate growth kinetic data for a fermentation process, to recover recombinant Pin1 protein in bacterial cell lysate using sonication and lastly, to quantify proteins using direct spectrometric and biolayer interferometry-based methods.