1. Cloning coding sequences of the parts into expression vector
a. Into Expression Vector
i. hCG Beta Subunit coding sequence was ligated into the pGEX expression vector
ii. hCG Beta 3 Loop coding sequence was ligated into the pGEX expression vector
b. Into pSB1C3 for submission
i. hCG Beta Subunit coding sequence was ligated into the pSB1C3 vector
ii. hCG Beta 3 Loop coding sequence was ligated into the pSB1C3 vector
We sent our DNA parts: hCG Beta Subunit coding part ligation into pGEX and the hCG Beta 3 Loop coding part into pGEX to Macrogen for sequencing and aligned the sequencing data against our expected constructs in SnapGene. The alignments matched, indicating a successful cloning of our coding parts into the pGEX vector.
2. Protein Expression and Isolation; hCG Antibody Recognition
a. Able to get the hCG Beta Subunit recognized by hCG antibodies
hCG Beta Subunit
The hCG beta subunit was expressed as a fusion with GST using the pGEX expression vector. After expression, and purification via glutathione column chromatography, the sample was tested for its ability to generate a positive signal on a ClinicalGuard Pregnancy Urine Test Strip. Although our test strips responded very clearly to 1.6 iU/L pure hCG purchased from Sigma-Aldrich (C1063-1VL), our recombinant fusion protein did not elicit a positive signal. We cleaved the fusion protein with HRV protease to remove the GST tag and visualized both the fusion protein and the cleavage product via Western blot using GST and hCG antibodies.
Figure 1.5 This blot contains protein elutions of the Beta 3 Loop-GST fusion protein, hCG Beta subunit-GST fusion protein, and the cleaved product of just hCG Beta subunit. This western blot was probed with anti-hCG antibodies. Lane 13 shows the hCG protein standard. Because this standard contains fully functional commercially available hCG, the band should be larger than the other recombinant hCG proteins in our other samples. The uncleaved and cleaved elutions of hCG (lanes 8 and 9) showed hCG protein. The Beta 3 Loop elutions did not contain any hCG Protein. However, the results from our coomassie blue stain leads up to think that our lysis step was not optimal, causing a large, aggregated protein mass in the cell debris (lane 6) from the expression and isolation process. The cell debris from hCG Beta subunit isolation did not contain a similar large mass, but rather produced correctly placed bands in the elutions.
We were able to successfully express the recombinant form of the hCG Beta subunit and an smaller protein which is an integral epitope present on the hCG Beta Subunit, the Beta 3 Loop. The hCG beta subunit was detected by the hCG antibodies which are similar to the non-specific polyclonal antibodies present in the Clinical-Guard Pregnancy Test strips we have used throughout the course of our project. However we did not get a positive response on the test strips with our purified protein. This could be due to very low concentrations and a lack of purity, there are still traces of other material in the elutions which could affect the antibody binding. The Beta 3 Loop was not lysed out of the cells properly, so this is just a matter of editing our experimental procedure.