In May, our team discussed potential project ideas and concluded that we wanted to continue last year’s project, which was focused on developing an endotoxin detection system to replace the horseshoe crab blood-based Limulus amoebocyte lysate assay (LAL). The proposed detection system would function as follows: Factor C is activated by the presence of endotoxin, this triggers autocatalysis releasing the hCG to be detected by a pregnancy test strip. Our plan was to generate the Factor C- hCG fusion protein expression construct via overlap extension PCR. We performed numerous attempts to fuse four segments of Factor C cDNA as shown below.
After weeks of following different protocols and using different primers, they all resulted in incorrectly sized bands on the gels, we still had been unsuccessful in producing a full-length Factor C coding sequence, much less the desired Factor C-hCG fusion. By mid-July, we had begun to contemplate dropping the idea of Factor C and focusing in on the hCG detection system part.
During this time, we also traveled to Baltimore, Maryland to participate in the 2018 Mid-Atlantic meet up. After presenting our project idea at the time, we received feedback and advice that helped shape our new project idea. We decided to form our project into something that reflected the essence of our lab at Georgia State: accessibility. We wanted to focus on the reporter aspect of our detection kit: hCG. hCG test strips are easily obtainable, easy to analyze with an all-or-nothing response, and cost-effective. Using hCG as a detection system would open up so many opportunities for all different kinds of lab spaces. From this point we aimed to produce a functional hCG protein fused to glutathione S transferase (GST), a protein widely used in fusion constructs to aid in purification and detection of desired synthetic proteins.
We set out to ligate our hCG coding part into the pGEX expression vector so that we could express the recombinant beta subunit hCG protein as a fusion with GST and test it on a pregnancy strip to make sure that hCG would work as a signal in a fusion protein.Over the counter pregnancy tests work by detecting the presence of hCG with antibodies. The antibodies, located within the fibrous end of the stick, contain enzymes with the ability to turn on dye molecules. When urine is applied onto the end of the stick it travels up and antibodies will then bind to the HCG, if it is present, and cause the enzymes to cleave activating the dye and causing a line to appear showing a positive result. To ensure the accuracy of the test, it contains a control line. The control line functions by appearing when the unbound enzymes flow through the stick and activate more creating a secondary line. If no secondary line is present then the test may be faulty. Our goal is to produce a synthetic hCG which can be added as a fusion protein to any other synthetic protein that serves as a component of a detection system. Activation of the detection system that results in mobilization of a previously immobilized fusion protein containing our hCG signal would then be detectable by a positive signal from an over the counter test strip.
We set out to ligate our hCG coding part into the pGEX expression vector so that we could express the hCG protein as a fusion with GST and test it on a pregnancy strip to make sure that hCG would work as a signal in a fusion protein.
We successfully inserted our hCG coding part into the pGEX vector and set about expressing and purifying the hCG-GST fusion protein At the beginning of August, we realized that we were still having a hard time getting our hCG-GST protein to function properly. Although we were seeing bands on our Western blots,when we tested our protein samples with test strips we got no reaction. The test strips gave a clear positive reaction when exposed to even tiny concentrations of commercially available hCG, but nothing with our purified samples. We decided to do additional research concerning producing an hCG-GST protein and found possible complications.
We found that hCG expressed in E. coli can form inclusion bodies that get stuck in the membrane and may impede proper purification and activity. Although we had been trying to synthesize the full beta chain of hCG, we read that smaller segments of the chain were sufficient to activate the hCG receptor. Perhaps small segments could also stimulate a positive signal on a pregnancy test strip while avoiding the problem of inclusion body formation. We decided to try producing a small segment of the beta chain, the beta 3 loop, which had been shown to bind to and activate the hCG receptor.
We designed a new coding part to express the hCG beta 3 loop. As before we wanted to express it as a fusion protein with GST to test its suitability as a signal in a fusion protein detector system. We successfully ligated the part into pGEX, proceeded with a protein expression and purification via glutathione column, and examined the results by SDS-PAGE with coomassie staining and Western blot probed with anti-hCG.