Overview of the ABOA 2025 project

Ten plasmids were designed and transformed into E. coli BL21(DE3)pLysS cells after synthesization. Thus, ten novel BL21(DE3)pLysS strains were made. Each of these strains were capable of periplasmically expressing one of the fusion proteins designed. These proteins were extracted from the periplasm with the osmotic shock method. After that, they were purified using the IMAC batch spin method. Throughout the extraction and purification processes, Bradford assays and SDS-PAGE were used to determine the success of the steps. Lastly, luminescence measurements were used to evaluate the functionality of the fusion proteins and to analyze the proof-of-concept for the VeriFied assay.

The VeriFied assay targets conformational changes in oxidized human serum albumin (HSA). Therefore, other methods like Ellman's assay and nanoDSF were used to study HSA oxidation. Using whole blood for Ellman’s assay would have given imprecise results because of hemoglobin and thus a protocol for lowering the amount of hemoglobin in whole blood was created. The samples handled according to these protocols were analyzed with different spectrophotometric measurements such as Bradford assay and measuring hemoglobin absorbance peaks.

Creating 10 novel BL21(DE3)pLysS strains for expressing fusion proteins

Ten constructs that are depicted on the Design page were designed. The DNA synthetization failed for eight of the ten plasmids after which sequence optimization was performed for the failed constructs. Order was placed for all the previously failed constructs in two different companies, one of which successfully synthesized all of the plasmids, and the other failed to synthesize seven plasmids of the eight ordered.

After the arrival of the expression-ready plasmids, transformation was conducted with the transformation protocol. The transformation was successful for nine plasmids in the first experiment (Figure 1). The transformation for the pET-IDT-C-His-Nb126-SmBit plasmid was unsuccessful on the first experiment, but was successful on the second. For the second experiment, higher amounts of DNA were used.

Figure 1. Successful transformation plates for (A) pET-IDT-C-His-NanoChuck, (B) pET-21(+)-Nb118-SmBit, (C) pET-21(+)-Nb80-LgBit, (D) pET-21(+)-Nb29-SmBit, (E) pET-21(+)-ALB8-LgBit, (F) pET-21(+)-Nb13-SmBit, (G) pET-IDT-C-His-Nb126-SmBit, (H) pET-IDT-C-His-Nb29-NanoLuc, (I) pET-21(+)-Nb80-NanoLuc, and (J) pET-21(+)-Nb77-SmBit. The plasmids with a pET-IDT C His backbone are plated on LB-agar plate with 50 µg/mL kanamycin and the plasmids with pET-21(+) are plated on LB-agar plate with 100 µg/mL ampicillin.

Due to successful transformations of all ten plasmids, ten novel E. coli BL21(DE3)pLysS strains that are capable of expressing novel recombinant fusion proteins were created (Table 1).

Table 1. The ten E. coli BL21(DE3)pLysS strains created during the ABOA 2025 project. Amp = ampicillin, CM = chloramphenicol, Kan = kanamycin.

Host organism	Plasmid	Fusion protein it can express	Antibiotic resistance
E. coli BL21(DE3)pLysS	pET-21(+)-ALB8-LgBit	ALB8-LgBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb29-SmBit	Nb29-SmBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb77-SmBit	Nb77-SmBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb80-NanoLuc	Nb80-NanoLuc	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb118-SmBit	Nb118-SmBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb13-SmBit	Nb13-SmBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb80-LgBit	Nb80-LgBit	Amp, CM
E. coli BL21(DE3)pLysS	pET-21(+)-Nb29-NanoLuc	Nb29-NanoLuc	Amp, CM
E. coli BL21(DE3)pLysS	pET-IDT-C-His-Nb126-SmBit	Nb126-SmBit	Kan, CM
E. coli BL21(DE3)pLysS	pET-IDT-C-His-NanoChuck	NanoChuck	Kan, CM

Summary of key results

• 10 novel recombinant fusion proteins designed
• 10 novel expression plasmids designed for expressing the proteins
• 10 novel E. coli BL21(DE3)pLysS strains created capable of expressing these proteins

Fusion protein expression, periplasmic extraction, and purification

Three iteratuons of protein expression, extraction, and purification were performed during the project. You can read about the related DBTL cycle here.

First iteration

The first iteration of protein expression and purification was done according to the first version of the expression, extraction, and IMAC purification protocol. In this iteration, the protocol was carried out with all ten proteins. Visual assessment of the Bradford assay indicated protein content for the first fraction of Nb77-SmBit (Figure 2). Second, third, fourth, and fifth fractions for all proteins contained no protein based on Bradford assay. For IMAC flow-throughs, only Nb29-NanoLuc seemed not to contain any protein, with Nb80-LgBit, and NanoChuck being unsure as the color was not clearly brown or blue.

Figure 2. Bradford assay for first, second and third fractions and flow-throughs performed on 11.7.25. A row contains the first fractions, B row the second fractions, C row the third fractions, and D row the flow-throughs. Column 1 contained Nb13-SmBit samples, column 2 and 12 MQ water, column 3 Nb80-LgBit, column 4 Nb77-SmBit, column 5 Nb80-NanoLuc, column 6 Nb29-NanoLuc, column 7 Nb29-SmBit, column 8 NanoChuck, column 9 Nb118-SmBit, column 10 ALB8-LgBit, and column 11 Nb126-SmBit. Nb118-SmBit flow-through was pipetted to E9 well instead of D9 well. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

Periplasmic extracts contained protein for all proteins (Figure 3). Periplasmic extracts for Nb80-NanoLuc, Nb29-NanoLuc, and NanoChuck, so all the fusion proteins with the full NanoLuc enzyme, seem to contain less protein than the other extracts.

Figure 3. Bradford assay for periplasmic extractions performed on 10.7.25. A1: Nb13-SmBit, A2: MQ water, A3: Nb80-LgBit (pipetting mistake), A4: Nb77-SmBit, A5: Nb80-NanoLuc, A6: Nb29-NanoLuc, A7: Nb29-SmBit, A8: NanoChuck, A9: Nb118-SmBit, A10: ALB8-LgBit, A11: Nb126-SmBit, A12: MQ water, B1: Nb80-LgBit. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 125 µL.

SDS-PAGE was performed for some of the most promising samples based on Bradford assay (Figure 4). A few samples that seemed to not contain any or only a little protein based on Bradford were included as well to be sure.

Figure 4. SDS-PAGE performed on 11.7. for first fraction and flow-through samples. The gel used was Mini-Protean TGX and it was run at 200 V for about 1 h. The tape at the bottom of the gel was forgotten to be removed. Gel was stained with PageBlueProtein Staining Solution (Thermo Fisher) and imagined with Gel Doc EZ Imager (Bio-Rad). The ladder was Precision Plus Protein Dual Color (Bio-Rad). The colored boxes indicate the area where the band for the protein should be. FT: flow-through, F1: first fraction.

With SDS-PAGE, the purifying success of Nb77-SmBit was verified. The faint slightly larger band in the lane could be Nb77-SmBit with the signal peptide still intact. A relatively large portion of Nb77-SmBit also seemed to not have bound to Co-NTA resin during purification as indicated by the band in the Nb77-SmBit flow-through lane. NanoChuck flow-through also seemed to contain NanoChuck protein that had not bound to Co-NTA resin. A similar issue might have occurred with Nb126-SmBit, Nb29-SmBit, Nb118-SmBit and ALB8-LgBit as well though it can not be conclusively said based solely on the gel as it ran a bit distorted because the tape was forgotten to be removed before the run. The first fraction of ALB8-LgBit seems to not contain any protein.

Summary of key results

• Nb77-SmBit extracted and purified successfully with IMAC

Second iteration

The second iteration at protein production, extraction and purification was done based on the second version of the expression, extraction, and IMAC purification protocol. Bradford assay was performed for all periplasmic extracts (Figure 5), flow-throughs (Figure 6), and all 3 fractions (Figure 7). All periplasmic extracts contained protein. Flow-throughs all contained protein, with Nb80-LgBit and NanoChuck containing only a little. None of the fractions contained any protein based on Bradford assay.

Figure 5. Bradford assay for periplasmic extractions performed on 31.7. C1: Nb29-NanoLuc, C2: Nb77-SmBit, C3: Nb80-LgBit, C4: NanoChuck, C5: Nb29-SmBit. The A row contains a BSA standard that was too concentrated. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

Figure 6. Bradford assay for flow-throughs performed on 31.7. A1: MQ water, A2: Nb29-SmBit, A3: Nb29-SmBit, A4: Nb77-SmBit, A5: Nb77-SmBit, A6: Nb80-LgBit, A7: Nb80-LgBit, A8: Nb29-NanoLuc, A9: Nb29-NanoLuc, A10: NanoChuck, A11: NanoChuck, A12: MQ water. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

Figure 7. Bradford assay for fractions performed on 31.7. Flow-throughs on A row are no longer readable. The readable version is depicted in Figure 7. B row contains the first fractions, C row the second fractions and D row the third fractions. All column 1 and 12 contain MQ water, column 2 and 3 contain Nb29-SmBit samples, columns 4 and 5 contain Nb77-SmBit samples, columns 6 and 7 contain Nb80-LgBit samples, columns 8 and 9 contain Nb29-Nanoluc samples and columns 10 and 11 contain NanoChuck. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

SDS-PAGE was used to analyze the periplasmic extractions of every protein, flow-throughs of Nb77-SmBit and Nb80-LgBit, lysates of Nb77-SmBit and Nb80-LgBit, and first fractions of Nb80-LgBit, Nb29-NanoLuc and Nb77-SmBit from this batch and also from the previous batch for reference (Figure 8).

Figure 8. SDS-PAGE performed on 1.8. for first fraction, periplasmic extract, and flow-through samples. The gels used were Mini-Protean TGX and they were run at 200 V for about 20 minutes before the voltage was changed to 150 V for about 5 minutes. Gels were stained with PageBlueProtein Staining Solution (Thermo Fisher) and imagined with Gel Doc EZ Imager (Bio-Rad). The ladder was Precision Plus Protein Dual Color (Bio-Rad). The colored boxes indicate the area where the band for the protein should be. FT: flow-through, PE: periplasmic extract, F1: first fraction.

Despite Bradford assay implying no protein content for Nb77-SmBit first fraction, SDS-PAGE showed a clear band of the correct size in the lane. The lane was more intense than the first fraction of Nb77-SmBit from 11.7., though it remains unsure if this is because of a more successful purification or because of the older fraction having degraded during storage. The band for Nb77-SmBit was also visible in the periplasmic extract. The flow-through for Nb77-SmBit did not seem to contain any protein of the correct size. These results seem to indicate that the resin recharging enhanced the purification results.

Periplasmic extracts for NanoChuck and Nb29-SmBit seemed to contain protein of the correct size. Nb29-NanoLuc and Nb80-LgBit extracts also seemed to contain protein of the correct size, but this seems to be of the same size of a native E. coli BL21(DE3)pLysS protein found in every lane. Thus, it is hard to say whether producing Nb29-NanoLuc or Nb80-LgBit was successful. First fractions for Nb29-NanoLuc and Nb80-LgBit did not contain any protein. Lysate samples analyzed for Nb77-SmBit and Nb80-LgBit contain many proteins of various sizes, which makes it hard to conclusively say if they contain the target protein. Nb80-LgBit flow-through has a faint band of correct size, so some of it could have been lost during purification.

Summary of key results

• Nb77-SmBit extracted and purified successfully with IMAC
• NanoChuck and Nb29-SmBit extracted successfully from the periplasm

Third iteration

The third iteration was done according to the third version of the expression, extraction, and IMAC protocol. Bradford assay was done on microtiter plates for periplasmic extracts (Figure 9), flow-throughs (Figure 10), and fractions (Figure 11). All periplasmic extracts contained protein, though Bradford assay indicated that the protein content was lower for the control strain BL21(DE3)pLysS that did not have any of the designed plasmids transformed in it. All flow-throughs contained proteins as well. None of the fractions had protein in them.

Figure 9. Bradford assay for periplasmic extracts performed on 14.8. Column 1 contains MQ water, column 2 ALB8-LgBit, column 3 Nb77-SmBit, column 4 Nb29-SmBit, column 5 Nb80-LgBit and column 6 control strain BL21(DE3)pLysS. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

Figure 10. Bradford assay for flow-through performed on 14.8. Column 7 contains MQ water, column 8 ALB8-LgBit, column 9 Nb77-SmBit, column 10 Nb29-SmBit, and column 11 Nb80-LgBit. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

Figure 11. Bradford assay for fractions performed on 14.8. A and B rows and C row wells from 1 to 3 contain a too concentrated BSA standard. MQ water is in wells C4-6. D row contains fractions for ALB8-LgBit, E row for Nb77-SmBit, F row for Nb29-SmBit, and G for Nb80-LgBit. Columns 1 to 3 contain the first fractions, columns 4 to 6 second fractions, and columns 7 to 0 third fractions. The wells contain 25 µL Protein Assay Dye Reagent Concentrate (Bio-Rad) and 10 µL protein sample in a final volume of 135 µL.

For every protein, the periplasmic extract and the first fraction were analyzed with SDS-PAGE (Figure 12). Additionally, the flow-throughs, second or third fractions for some of the proteins were analyzed so that all proteins had at least 3 samples loaded to the gel. Periplasmic extract of the control strain BL21(DE3)pLysS was also analyzed.

Figure 12. SDS-PAGE performed on 15.8. for samples of Nb29-SmBit, Nb77-SmBit, ALB8-LgBit, control strain BL21(DE3)pLysS, and Nb80-LgBit. The gels used were Mini-Protean TGX and they were run at 150 V for about 10 minutes before the voltage was changed to 125 V for about 50 minutes. Gels were stained with PageBlue Protein Staining Solution (Thermo Fisher) and imagined with Gel Doc EZ Imager (Bio-Rad). The ladder was Color Prestained Protein Standard (New England Biolabs). The colored boxes indicate the area where the band for the protein should be. The calculated protein size is in brackets after the sample name. PE: periplasmic extract, FT: flow-through, F1: first fraction, F2: second fraction, F3: third fraction.

The periplasmic extracts for Nb29-SmBit and Nb80-LgBit had bands of correct sizes, indicating a successful expression and extraction of these proteins. While the periplasmic extracts for Nb77-SmBit and ALB8-LgBit contain protein in the area where the proteins should be, there are no distinct bands indicating a presence of the fusion proteins. SDS-PAGE verified no protein in the fractions as had already been indicated by Bradford assay. The flow-through of Nb29-SmBit seems to contain Nb29-SmBit which means it did not bind to the Co-NTA resin. The control strain periplasmic extract has a band of about 40 kDa that also appears in all of the periplasmic extracts. Thus, it has now been verified as a native BL21(DE3)pLysS protein.

Summary of key results

• Nb29-SmBit and Nb80-LgBit extracted successfully

Luminescence signal measurement to measure the function of the fusion proteins

To determine the background signal produced by the single fusion proteins with only a fragment of the NanoLuc enzyme and to confirm that the majority of the signal was due to successful enzyme fragment complementation of the NanoLuc enzyme, the signals of individual assay components were measured. The expectation was that the signal produced by these controls would be low enough compared to the signal from tests using either both enzyme fragments or the whole NanoLuc enzyme to confirm successful function of NanoLuc.

Background signal caused by MQ water, TSE (200 mM Tris-HCl pH 7.5; 500 mM sucrose, 1 mM EDTA), the buffer used to extract the proteins from the periplasm, HSA, and the control strain with and without HSA were measured (Table 2).

Table 2. Background signal of MQ water, HSA, and TSE (200 mM Tris-HCl pH 7.5; 500 mM sucrose, 1 mM EDTA) buffer controls and control strain E. coli BL21(DE3)pLysS. Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained either 40 µL of TSE, 1 µg human serum albumin (HSA), 10 µL periplasmic extract of E. coli BL21(DE3)pLysS, or 80 µL of MQ water, 0.02 M phosphate buffer pH 8 and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 (HSA) or 100.5 (TSE, MQ, control strain) µL. Variance reported in ± SD of three technical replicates.

Sample	Highest luminescence reading recorded (RLU) ± SD
TSE	209 ± 29
MQ water	238 ± 68
1 µg HSA	986 ± 246
E. coli BL21(DE3)pLysS	513 ± 172
E. coli BL21(DE3)pLysS with 1 µg HSA	332 ± 79

All luminescence measurements were performed with periplasmic extracts, because only Nb77-SmBit purification was successful. The background signal caused by the individual fusion proteins, containing only one enzyme fragment, Nb80-LgBit, Nb77-SmBit, and Nb29-SmBit were measured (Figure 13). The individual proteins produce a maximum bioluminescence signal of approximately 16 000 RLUs.

Figure 13. Luminescence signal produced by different volumes of periplasmic extractions of Nb80-LgBit (a), Nb29-SmBit (b), and Nb77-SmBit (c). Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained either 70, 50, 30, 20, 10 or 5 µL of periplasmic extract, 0.02 M phosphate buffer pH 8 and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 µl. The signal for t=0 min was measured when the substrate had not been added to the wells. The fusion proteins used were extracted on 31.7. Variance reported in ± SD of three technical replicates. Measurements were performed on 8.8. The Y axis has been scaled to highlight the difference between the luminescence signal produced by proteins with only a fragment of the enzyme and the pair that can form a functional enzyme.

The luminescence signal produced by Nb80-LgBit and Nb29-SmBit as well as Nb80-LgBit and Nb77-SmBit pairs without HSA were measured at multiple time points to determine the peak values (Figure 14). For the Nb80-LgBit and Nb29-SmBit pair, the peak seems to be between 45-60 minutes. For the Nb80-LgBit and Nb77-SmBit pair, the peak seems to be between 60-75 minutes.

Figure 14. Luminescence signal produced by Nb80-LgBit and Nb29-SmBit pair (A) and Nb80-LgBit and Nb77-SmBit pair (B) without HSA. Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained 10 µL of periplasmic extracts for both proteins of the pair, 0.02 M phosphate buffer pH 8 and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 µl. The fusion proteins used were extracted on 31.7. Variance reported in ± SD of three technical replicates. Measurements were performed on 8.8.25. HSA: human serum albumin.

With HSA present, the signal is lower for both of the pairs (Figure 15). This could be because of HSA binding the complementary proteins too far from each other to form a functional enzyme thus decreasing the luminescence signal.

Figure 15. Luminescence signal produced by Nb80-LgBit and Nb29-SmBit pair (a) and Nb80-LgBit and Nb77-SmBit pair (b) with HSA. Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained 20, 10 or 5 µL of periplasmic extracts for both proteins of the pair, 0.02 M phosphate buffer pH 8, 1 µg HSA, and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 µL. For the Nb80-LgBit and Nb29-SmBit pair, the Nano-Glo luciferase assay reagent used contained 2 % of Nano-Glo® Luciferase Assay Substrate in the Nano-Glo® Luciferase Assay Buffer while the reagent used with Nb80-LgBit and Nb77-SmBit pair contained 1 % of substrate in the buffer. The signal for t=0 min was measured when the substrate had not been added to the wells. The fusion proteins used were extracted on 31.7. Variance reported in ± SD of three technical replicates. Measurements were performed on 8.8.25. HSA: human serum albumin.

NanoChuck has both of the NanoLuc enzyme fragments separated by a linker, whereas Nb29-NanoLuc has the functional NanoLuc enzyme connected to Nb29 with a linker. Thus, it was hypothesized that the non-split enzyme would produce more luminescence. While Nb29-NanoLuc does produce more luminescence than NanoChuck, the signal increases when volume decreases (Figure 16). It seems that perhaps the wells that should contain 5 µL of Nb29-NanoLuc contain 20 µL of Nb29-NanoLuc because of a pipetting mistake. This mistake went unnoticed while pipetting but it seems like a logical explanation.

Figure 16. Luminescence signal produced by Nb29-NanoLuc (a) and NanoChuck (b). Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained 20, 10 or 5 µL of periplasmic extracts, 0.02 M phosphate buffer pH 8 and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 µl. The Nano-Glo luciferase assay reagent used contained 1 % of Nano-Glo® Luciferase Assay Substrate in the Nano-Glo® Luciferase Assay Buffer. The signal for t=0 min was measured when the substrate had not been added to the wells. The fusion proteins used were extracted on 31.7. Variance reported in ± SD of three technical replicates. Measurements were performed on 8.8.25.

With these luminescence measurements, the presence of luminescent proteins in the periplasmic extracts done on 31.7. for Nb80-LgBit, Nb77-SmBit, Nb29-SmBit, NanoChuck, and Nb29-NanoLuc was confirmed. The luminescence signal indicates that the NanoLuc fragments had folded functionally.The luminescence signal also implies Nb29-NanoLuc and Nb80-LgBit having expressed even though it could not be conclusively stated based on the gel.

The proteins containing only a fragment of NanoLuc produced only background signal when compared to the signal produced by the pairs. The luminescence signal produced by proteins containing the full NanoLuc enzyme produced more signal than the pairs as is to be expected. However, comparing the signals produced by the proteins can not conclusively be done as they were periplasmic extracts and thus the precise concentrations of the proteins remains unknown.

Four different pairs were used with extracted bloodstains of different ages to see if the signal would decrease as the bloodstain got older as had been hypothesized when designing VeriFied (Figure 17). The periplasmic extracts extracted on 14.8. were used. Based on SDS-PAGE, these periplasmic extracts might not contain Nb77-SmBit or ALB8-LgBit.

Figure 17. Bloodstain age measurements. Luminescence signal was measured 50-60 minutes after adding Nano-Glo luciferase assay reagent. Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained 10 µL of periplasmic extracts for both proteins, 10 µL of extracted bloodstain (ages 1, 2, 3, 4, 5, 8, 11, 15, 22 or 29 d), 0.02 M phosphate buffer pH 8 and 0.5 µl Nano-Glo luciferase assay reagent in a final volume of 101 µl. The fusion proteins used were extracted on 14.8. Variance reported in ± SD of three technical replicates except Nb77-SmBit and Nb80-LgBit pair had only 2 replicates for 1 d. Measurements were performed on 22.8.

The Nb77-SmBit and Nb80-LgBit produces a high luminescence signal indicating a successful enzyme fragment complementation. The luminescence signal seems to decrease from day 1 to 5 before plateauing. The signal begins to decrease after day 15 until day 22. After day 22, the signal seems to begin to increase, though it can not be said conclusively as there are no older bloodstains tested than of 29-days-old. There is a sudden drop in signal on day 3 in all three replicates. The outlier is likely an error in sample preparation or measurement, but the cause of this drop would need to be verified in future studies.

The Nb29-SmBit and ALB8-LgBit pair only produced background signal, with the highest measured signal being 364 RLU with a standard deviation of 21. This signal is comparable to the signal produced by MQ water or, for example, the control strain, and it is much lower than the signals previously measured from single proteins with only one enzyme fragment. Thus, it seems likely that the periplasmic extracts for ALB8-LgBit and Nb29-SmBit do not contain any of the fusion proteins, only contain very small amounts or the proteins failed to fold correctly.

Nb29-SmBit and Nb80-LgBit and Nb77-SmBit and ALB8-LgBit pairs also produce low luminescence signal when compared to the Nb77-SmBit and Nb80-LgBit pair. As they still produce a little signal, the signal could be caused by a single fusion protein of either Nb77-SmBit or Nb80-LgBit. The concentrations could also just be too low or the proteins may have folded incorrectly. With these pairs, a similar correlation can be seen between the luminescence signal and the bloodstain age as with the higher luminescence signal producing pair Nb77-SmBit and Nb80-LgBit. From day one to day five, the luminescence signal seems to steadily decrease before plateauing before day 15 when the signal will begin to drop again. After day 22, the luminescence signal seems to increase.

Based on previous measurements indicating that the presence of HSA affects luminescence readings, preliminary optimization trials were done to find the correct ratio between HSA and fusion proteins. All pairs were measured with two different amounts of HSA (Figure 18).

Figure 18. Human serum albumin (HSA) concentration’s effect on the luminescence signal produced by four different pairs. Luminescence signal was measured 50-60 minutes after adding the Nano-Glo luciferase assay reagent. Measurements were performed with Hidex Sense Microplate reader on a white Nunc Maxisorp 96-well plate. The wells contained 10 µL of periplasmic extracts for both proteins, 0.25, 0.5, 1 or 2 µg HSA, 0.02 M phosphate buffer pH 8 and 1:200 Nano-Glo luciferase assay reagent in a final volume of 100.5 µL. The final volume was 103.5 µL for Nb80-LgBit and Nb29-SmBit with 2 µg HSA. Periplasmic extracts were extracted on 14.8. Measurements were performed on 22.8.

With all the tested pairs and HSA concentrations, it is clear that the HSA concentration affects the luminescence signal. However, the magnitude of the change and whether it decreases or increases as HSA concentrations change, varies between pairs. Most likely all pairs have an optimal HSA concentration, which can not be conclusively stated based on these findings as only two HSA concentrations per pair were tested.

Summary of key results

• The NanoLuc fragments have folded correctly in Nb80-LgBit, Nb77-SmBit, Nb29-SmBit, NanoChuck, and Nb29-NanoLuc
• The proteins with only a fragment of the NanoLuc enzyme only produce background signal when compared to pairs
• HSA affects the signal levels produced by pairs
• The bioluminescence signal produced by the Nb77-SmBit and Nb80-LgBit pair decreases as bloodstains get older
• The bioluminescence signal produced by the Nb77-SmBit and ALB8-LgBit pair an the Nb29-SmBit and Nb80-LgBit pair decreases as bloodstains get older though the signal is much lower

Studying HSA oxidation with nanoDSF and Ellman’s assay

Nano differential scanning fluorimetry (nanoDSF) and dynamic light scattering (DLS) were used to analyze the conformational stability of two different HSA samples, one more reduced than the other (Figure 19). The less reduced sample is untreated HSA (Sigma-Aldrich), while the more reduced sample is HSA (Sigma-Aldrich) that has been treated with dithiothreitol (DTT). The aim of the analysis was to see whether HSA’s oxidation state affected its stability as well as to see whether HSA would stay intact at 60 °C, the temperature used in the final HSA-hemoglobin separation protocol.

Figure 19. Conformational stability comparison of human serum albumin (HSA) and reduced HSA using nano differential scanning fluorimetry (nanoDSF) and dynamic light scattering (DLS). Two technical replicates of each sample were measured. Purple sample is pure HSA (≥99 % Sigma-Aldrich) (15 µM) in phosphate buffered saline (PBS). Green sample is HSA (≥99 % Sigma-Aldrich) (15 µM), incubated for 5 min in PBS with 10.8x molar excess of dithiothreitol (DTT) (162 µM) in room temperature before desalting with Amicon Ultra Centrifugal Filter 0.5mL 30K MWCO. nanoDSF and DLS were measured with NanoTemper Prometheus Panta. The onset of unfolding or size increase (T_on) indicates the temperature at which the protein starts to unfold or increase in size. The melting point (T_m) of the samples indicates the temperature at which 50 % of the proteins in the sample have unfolded. a. Ratio of absorbances measured at 350 nm / 330 nm that indicates the change in environment of tryptophan as a function of time. b. First derivative of the ratio of absorbance readings measured at 350 nm / 330 nm. c. DLS measurement that indicates the cumulative radius of the particles in the sample as a function of time.

The observed difference in melting point (T_m) of 0.99 °C, along with a slight difference in measured cumulative radius and onset of size increase difference of (T_on) 2.24 °C measured between the HSA and reduced HSA samples point towards slight structural differences between the samples. The results further corroborate oxidation causing structural changes in HSA, although additional measurements with more drastically oxidized or reduced samples would likely show a more substantial difference. The direction of change toward a more thermally stable conformation in the oxidized HSA is consistent with previous studies pointing toward oxidized HSA having a more thermally stable structure [1]. Even though the measured differences are minor, they prove that the method can be used to study how oxidation affects the conformational stability of HSA. At 60 °C, HSA is still intact meaning the heat treatment used in the separation protocol does not denaturate HSA.

Additionally, Ellman’s assay was used to study HSA oxidation. For performing measurements with Ellman’s assay, a cysteamine standard was made (Figure 20). Ellman’s assay was envisioned to be used for quantifying the oxidation states of the untreated and reduced HSA samples analyzed with nanoDSF and DLS. However, due to technical errors during the measurements,reliably quantifying the oxidation difference between the samples can not be done. Despite this, Ellman’s assay showed a difference between the samples, implying that there is a higher percent of reduced Cys34 in the samples treated with DTT. Even though the results are not conclusive, they indicate that the method is suitable for studying HSA oxidation, and would likely have provided more precise results with further optimization.

Figure 20. Ellman’s assay standard curve using cysteamine. In a final volume of 100 µl, 5 µL of 1 mM Ellman’s Reagent solution was reacted with 5 µL cysteamine solution of 0.2-1.0 mM. The reagents were dissolved into 0.1 M phosphate buffer pH 8.0, which was also used as the reaction buffer. The samples were measured with Tecan Infinite 200 Pro plate reader after incubating at room temperature for 15 minutes. Variance reported in ± SD of three technical replicates.

Summary of key results

• nanoDSF, DLS, and Ellman’s assay seem to be promising methods to study HSA oxidation.
• Preliminary measurements using nanoDSF and DLS show structural differences between HSA at different oxidation states, although further testing is required to confirm the results.

HSA purification from dried bloodstains for HSA oxidation measurement

Purification of HSA from dried bloodstains was done with the goal to measure HSA oxidation over time in aged bloodstains. The measurements were also done to evaluate the protocol's success. HSA purification was performed with the "Final Hemoglobin-HSA Separation protocol". After the blood extraction samples were handled the way in the protocol, spectrophotometric measurements were used to examine different protein concentrations. Total protein concentrations were measured using Bradford assay, hemoglobin concentration with a spectrophotometric method and SDS-PAGE was run with blood extractions and heat purified samples. Bromocresol green measurements were also done, but reliable and reproducible results could not be produced with that method. You can read more about bromocresol green measurements on the Engineering page.

Spectrophotometric measurements for detecting hemoglobin in purified bloodstain samples

Spectrophotometric measurements were performed for the different aged bloodstain heat purified samples to study the hemoglobin concentrations on 20.8.2025. (Figure 21). Dilution ratio for measuring the heat purified samples was 1:128. According to the hemoglobin measurements, the hemoglobin concentrations were lower in the older bloodstain heat purified samples. A negative trendline can be observed from the values.

Figure 21. Hemoglobin measurements performed on 20.8. for different aged bloodstain heat purified samples. The samples were measured at 420 nm with Perkin Elmer Lambda Bio 40 UV/VIS spectrometer. Bloodstain ages vary from 1 day to 29 days old bloodstains. Variance reported in ± SD of four technical replicates.

Summary of key results

• Hemoglobin was quantified from the different aged bloodstain heat purified samples

Bradford measurements for defining total protein concentrations in purified bloodstain samples

Bradford measurements were performed for the different aged bloodstain heat purified samples to study the total protein concentrations in samples on 22.8.2025. (Figure 22). The dilution ratios of the heat purified samples were 1:128. The total protein concentrations are lower in the older bloodstains which can be seen when a trendline is fitted onto the values.

Figure 22. Bradford measurements performed on 22.8. for different aged bloodstains heat purified samples. The samples were measured at 595 nm with Tecan Infinite 200 Pro plate reader. The bloodstain ages vary from 1 day to 29 days old bloodstains. Variance reported in ± SD of four technical replicates.

A standard curve was also made for Bradford measurements using bovine serum albumin (BSA). (Figure 23) The earlier mentioned total protein concentrations were outside of this standard curve as the measured total protein concentrations were higher than the highest standard sample, 0.5 mg/mL.

Figure 23. Bradford standard curve made with bovine serum albumin (BSA). The standard samples were measured at 595 nm with Tecan Infinite 200 Pro plate reader. The concentrations of the measured standard samples were between 0.0 and 0.5 mg/mL. Variance reported in ± SD of three technical replicates.

Summary of key results

• Total protein concentrations were quantified from the different aged bloodstain heat purified samples. The samples were outside of the standard curve in the end.

SDS-PAGE for blood samples to assess purification success

The heating purification protocol’s (Final Hemoglobin-HSA separation protocol) effect was confirmed by performing an SDS-PAGE of different aged bloodstain extraction and heat purified samples (Figure 24). Dilution ratios for the extraction samples were 1:100 and for the heat purified samples 1:2. The heat purified samples were overloaded on the gels. Bands corresponding to the size of HSA and hemoglobin monomers can be detected on the gels. As already confirmed in the Prometheus Panta measurement, heating the sample to 60 ℃ does not cause HSA to irreversibly unfold past molten globule state, which is further supported by the SDS-PAGE. The heat purification can be seen as successful as the HSA band can visually be seen on the gels and has not been broken down into smaller fractions. Still, the impact of the purification on HSA conformational changes and its oxidation state remain unclear with these results and would need further research.

Figure 24. SDS-PAGEs performed on 26.8. for different aged bloodstain extraction and heat purified samples. (a) Gel A contained bloodstain extractions and heat purified samples from 1 day to 5 days old bloodstain samples. (b) Gel B contained bloodstain extractions and heat purified samples from 11 days to 29 days old bloodstain samples. The gels used were Mini-Protean TGX and were run at 150 V for about 50 minutes. Gel was stained with PageBlueProtein Staining Solution (Thermo Fisher) and imagined with Gel Doc EZ Imager (Bio-Rad). The ladder was Precision Plus Protein Dual Color (Bio-Rad). The colored arrows indicate the area where the band for the HSA and hemoglobin monomers should be. xdFE(A or B) = x days old bloodstain extraction, xdFAS = x days old bloodstain heat purified sample.

Summary of key results

• The heat purification protocol seems to be successful so far. Its effect on HSA conformational changes and oxidation state is still uncertain and needs to be confirmed in the future.

References

[1] G. Sancataldo, V. Vetri, V. Foderà, G. Di Cara, V. Militello, and M. Leone, “Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation,” PLoS ONE, vol. 9, no. 1, p. e84552, Jan. 2014, doi: https://doi.org/10.1371/journal.pone.0084552.