EHSBNUPlasmid design
To produce recombinant tachypleus tridentatus clotting FG, we designed five plasmids in total for separate expression of its alpha and beta subunits as well as their co-expression.
For the E.Coli expression system, we designed two pET-28a plasmids with the optimized sequences of FG alpha and beta subunits respectively. In addition, we designed a pET-Duet1 plasmid with both sequences so that the subunits may bind more easily after expression. We hoped to accommodate this eukaryotic protein into a prokaryotic expression system, whether in selecting only the mature peptide sequences of the subunits from NCBI GenBank, or in performing codon optimization with NovoPro ExpOptimizer for elevated expression efficiency.
Since previous studies on recombinant FG production all employed eukaryotic expression hosts, we also designed two eukaryotic plasmids in case the protein failed to properly express through E.Coli. We optimized the sequences for sf9 insect cell expression hosts, then inserted them into two Pfastbac1 plasmids.
Protein expression with E.Coli BL21
We transformed the pET-28a plasmids into E.Coli BL21(DE3) cells through heat shock, and inoculated the bacterial culture into kanamycin LB liquid medium at a concentration of 1:4. The bacterial culture was incubated at 37 ° C and 200 rpm for 1.5 hours before sample optical density was measured. When OD fell between 0.6-0.8, we induced protein expression with IPTG at a concentration of 1:1000.
However, during subsequent performance of SDS-PAGE, we discovered that the two subunits failed to express. No prominent protein bands were detected at 72 Kb and 37 Kb, corresponding to the alpha and beta subunits respectively. Our western blot assay similarly yielded no results.
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Image 1: SDS-PAGE & WB of failed expression
To investigate why neither subunits were expressed, we employed differents strain of competent cells, including Rosetta, Origami, and SHuffle, and repeated the previous experimental procedures. Target protein bands were obserservable at their corresponding molecular weights, showing ameliorated protein expression.
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Image 2: SDS-PAGE of successful expression
We then performed mass spectrometry to more accurately identify and quantify the expressed subunits. The protein bands were destained and dithiothreitol was added to break the disulfide bonds. We then enzymatically incised the protein into fragments of 700-3000 amino acids to facilitate mass spectrometry assay. The results indicated successful expression of both the alpha and beta subunits, though at a relatively low level. To boost production level, we further expressed the subunits with 5 liters of E.Coli. The mass expression and purification procedures are currently progress, and successful results are anticipated within a week.
Experimental plans with sf9 insect cells
Since prokaryotic expression hosts may lack the protein modification mechanisms required for producing a fully functional horseshoe crab protein, we also conducted experiments with Spodoptera frugiperda 9 insect cells.
BDG assay system construction
Upon acquiring the purified protein, we hope to test the activity of our protein, and the efficiency of recombinant Factor G in fungal infection detection. In the natural 1,3-BDG coagulation pathway, upon binding with fungal cell walls and detecting (1→3)-β-D-glucans (BDG), the α subunit of Factor G hydrolyses, releasing the β subunit, triggering a gelatinization/coagulation cascade, immobilizing fungal antigens[1]. In synthetic coagulation pathways, an indicator of this process is the hydrolysis of a synthetic chromogenic substrate, Boc-TGR-pNA(Nα-t-butyloxycarbonyl-L-threonyl-glycyl-arginine-p-nitroanilide), by the β subunit of Factor G, which releases yellow p-nitroaniline. We can then quantitatively measure the intensity of the color by spectrophotometrically measuring absorbance at 405nm wavelength.
We plan to perform a partial reconstruction of the pathway by first qualitatively testing both recombinant subunits separately. We will add Boc-TGR-pNA to our purified β subunit protein, and watch for qualitative changes in color that indicate a chromogenic reaction, and confirms the activity of our recombinant β subunit. We will then add (1→3)-β-D-glucans to purified α subunit protein, allowing it to react thoroughly, and running samples with SDS-PAGE to visualize whether the α subunit has undergone activation via proteolytic cleavage.
Subsequently, we plan on quantitatively testing the activity of our recombinant β subunit quantitatively measuring absorbance at 405nm, creating a standard curve, and comparing it to a standard curve created by current commercial G test kits. Finally, we will test the quantitative efficacy of our combined α and β subunits obtained by tandem expression. By comparing the efficiency of the BDG-assay-system using our recombinant FG protein with existing BDG test kits made from horseshoe crab protein, we hope to test the efficacy of our reconstructed fungal detection system alongside its ecological merits.