During my master’s degree at the University of Massachusetts Lowell, I participated in a laboratory course focusing on the production, isolation, purification, and characterization of DNA polymerase from Thermus aquaticus (“Taq”). Over the course of the semester, I performed an entire lab-scale biological production campaign for this product:
A cell culture of E. coli, genetically modified with the addition of the pAKTaq plasmid, was initiated and grown for several cell cycles. Once the cell growth plateaued, pAKTaq expression was induced with IPTG, an allolactose analogue.
After allowing the cell culture to express pAKTaq overnight, the cells and media were separated by centrifugation, discarding the supernatant (as all Taq DNA polymerase remains within the cells).
The bacterial pellet was resuspended in buffer, and the cells lysed with a lysozyme. This created the raw harvest.
Taq DNA polymerase is stable at 75 °C, while most E. coli proteins are not. By adding PMSF (a serine protease inhibitor) and heating the raw harvest to 75 °C, most E. coli proteins denatured and precipitated out of solution. The raw harvest was then centrifuged, and the pellet discarded.
PEI is a positively charged polymer that binds to negatively charged proteins, causing them to precipitate. This phenomenon can be used to separate proteins based on pI value (the pH at which the net charge on the protein is zero). By varying the pH of the solution, proteins can be selectively targeted for precipitation with PEI.
PEI was added to the harvest, causing the Taq DNA polymerase to precipitate out of solution. The harvest sample was centrifuged, and the supernatant discarded.
The pellet, containing PEI and Taq DNA polymerase, was washed with a low-salt solution, allowing other bound impurities to re-dissolve. This mix was centrifuged again, and the supernatant discarded.
The pellet was then washed with a high-salt solution, allowing the Taq DNA polymerase to re-dissolve. This mix was centrifuged again, and the pellet discarded.
To reduce the salt in the sample (from the previous step) and prepare it for chromotography, the sample was subjected to buffer exchange by dialysis. The sample was placed within in a semipermeable membrane, and left overnight in a low-salt bath. The salt concentration equalized across the membrane.
To remove the last of the impurities from the sample, batch cation exchange chromatography with CM-Sephadex resin was carried out. The proteins within the sample were allowed to bind to the CM-Sephadex resin under very-low-salt conditions. Impurities were eluted with a low-salt wash. Finally, the Taq DNA polymerase was eluted with a higher-salt
After chromatography, the Taq DNA polymerase sample was at a very low concentration, dissolved in about 25 mL of water. The sample was concentrated with centrifugal ultrafiltration. The sample was placed in an ultrafiltration vial, and centrifuged. Water (from the sample) permeated the ultrafiltration membrane to fall to the bottom of the vial, while Taq DNA polymerase was retained at the top. Following ultrafiltration, the sample volume was reduced to about 1 mL, with about 24 mL of water having been removed.
Four methods were used to evaluate final protein concentration:
Each method was performed against control samples with known concentrations of bovine serum albumin, with the resulting absorbances measured by spectrophotometry. The control samples created a curve to define a relationship between concentration and absorbance.
To measure the molecular weight of the target protein, a sample of Taq DNA polymerase was combined with detergent sodium dodecyl sulfate (SDS) and heated to denature the proteins. This sample was run in a polyacrylamide gel electrophoresis (PAGE) alongside a blank control and a sample of commercially available Taq DNA polymerase.
The gel was then stained with Coomassie Brilliant Blue, and the proteins transfered to nitrocellulose paper by electrophoresis. By washing the nitrocellulose paper with primary and secondary antibodies attached to a chemiluminescent marker, a Western blot image was obtained. By comparing the band representing laboratory-produced Taq DNA polymerase against the band representing commercially available Taq DNA polymerase, target protein identity could be verified.
Another sample of laboratory-produced Taq DNA polymerase was used in a polymerase chain reaction (PCR) procedure. As with the SDS-PAGE gel, this was also done alongside a control blank sample and a sample of commercially available Taq DNA polymerase.
The PCR was run for 30 cycles, allowing for replication of the initial DNA primer strands.
An agarose gel was casted. The three DNA samples were dyed with GelGreen DNA stain and run in the gel. By comparing band placement in the DNA replicated with laboratory Taq DNA polymerase, to band placement in the DNA replicated with commercial Taq DNA polymerase, enzyme activity could be verified.
This course allowed me to develop hands-on experience with all stages of biological protein production at the laboratory scale, and equipped me for similar work in a professional context.