Introduction
In this page, we summarised the DNA cloning, transformation, and protein expression results from the Engineering Page. For information on testing the functionality of our expressed fusion proteins, please visit the Proof of Concept Page.
Chromoproteins
Tinsel Purple (tsPurple) & Nanobody-tsPurple
Purple colours were observed in the elution samples of both tsPurple and Nanobody-tsPurple. As shown below in Figure 1. This suggests that tsPurple is present in both protein samples.
Figure 1: Purification picture of nanobody-tsPurple and elution samples of tsPurple
For tsPurple, the concentration of protein samples ranged from 522.67 μg/mL to 15353.33 μg/mL depending on depending on the eluted order. For nanobody-tsPurple, the expressed protein concentrations were 35.92 μg/mL.
However, with further analysis of the SDS-PAGE, we observe no band around the size of nanobody-tsPurple (41.2 kDa), shown in Figure 2. Therefore, it is likely that tsPurple was expressed without nanobody fusing to it. This might be due to the breakage of the linker between nanobody and chromoprotein during protein periplasmic secretion. Because the His-tag was attached to the C terminus of chromoprotein, only the chromoprotein was purified. The faint lilac colour can be then explained by expressing the retained tsPurple insert in the plasmid. Due to the timeframe of the project, we were unable to verify this hypothesis. Since the nanobody section was not expressed for nanobody-tsPurple, we did not carry out ELISA to test out the affinity of the conjugate.
Figure 2: SDS-PAGE result of nanobody-tsPurple with no band of correct size showing.
gfasPurple & Nanobody-gfasPurple
Since gfasPurple and tsPurple are very similar in their functionality as a detection protein (exhibiting colours that can be visualized by the naked human eye) and tsPurple exhibited better cloning and expression performance, we decided not to express any of the gfasPurple conjugates. Progress paused after the plasmids were successfully cloned.
Single NanoLuc
NanoLuc & Nanobody-NanoLuc
For the single NanoLuc conjugates, the correct proteins were expressed and purified. The length of the protein was verified with a SDS-PAGE (Figure 3). From the SDS-PAGE results, a band was observed in lane 1 at about 20 kDa which matches with the expected size of NanoLuc (19.1 kDa). For nanobody-NanoLuc, lane 2, a band was observed at about 35 kDa, consistent with the expected size of 34.8 kDa. From Bradford assay, the concentration of nanobody-NanoLuc was 5.05 μg/mL, and the concentration of NanoLuc was 236.75 μg/mL.
Figure 3: SDS-PAGE result of NanoLuc (lane 1) and nanobody-NanoLuc (lane 2).
Double NanoLuc
NanoLuc-NanoLuc
The NanoLuc-NanoLuc construct was successfully cloned into DH5α with a gel extraction step after a PCR process to remove any extra bands. We found out that gel extraction is more efficient in eliminating incorrect amplicon comparing to a PCR cleanup. Due to time constrain, no further cloning or protein expression was done for NanoLuc-NanoLuc.
Nanobody-NanoLuc-NanoLuc
From SDS-PAGE, a dark band was observed at about 60 kDa which is slightly larger than the length of nanobody-NanoLuc-NanoLuc (54.7 kDa) (Figure 4). This minor difference is normal because SDS-PAGE is often associated with some degree of inaccuracy. From Bradford assay, the concentration of nanobody-NanoLuc-NanoLuc elution samples were determined to be ranging from 182.58 μg/mL to 944.25 μg/mL depending on the eluted order.
Figure 4: SDS-PAGE result of nanobody-NanoLuc-NanoLuc.