Part:BBa_K5301000

MSP1E3D1 is a genetically engineered protein, which mimics the function of ApoA-I.

Introduction

The goal of BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Our parts collection can be mainly divided into two categories: mono-MSPs that could construct small or large nanodiscs through self-cyclization, and large cyclic MSP formed by the interaction and linkage of multiple MSPs, which are used for constructing large nanodiscs. They are closely linked together due to their common function of manufacturing nanodiscs.

Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs. We further sought and obtained spNW15 and spNW50 that utilized the automatic covalent linkage of SpyTag and SpyCatcher to enhance the cyclization efficiency and enable the automatic cyclization of MSP, in order to manufacture nanodiscs of different diameters more simply. On this basis, taking NW15 as the basic component, we designed the multi-polymerized MSP, consisting of three linear MSP monomers. Only when three mono-MSPs interact with each other can they form cyclized MSP and achieve their function of constructing nanodiscs. It provides a more flexible solution for manufacturing large nanodiscs, while reducing the expression pressure on the chassis bacteria and avoiding the difficulty of purifying large proteins.

This Part Collection aims to provide a series of easily accessible and distinctively characterized MSP proteins as a toolkit for the assembly of nanodiscs. Users can easily select which MSP to produce and utilize based on their own needs to manufacture nanodiscs. The nanodiscs fabricated using the MSP we designed can be used for stabilizing amphipathic proteins, studying the structure and function of amphipathic proteins, drug delivery, developing novel antiviral drugs, etc., and possess broad application prospects.

This part produces MSP1E3D1, fabricating nanodiscs with relatively high lipid fluidity.

Usage and Biology

Nanodisc technology is a widely applicable approach to render membrane proteins soluble in aqueous solutions in a native-like bilayer environment, where the membrane proteins remain stable and active. The Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein (MSP), a genetically engineered protein, which mimics the function of Apolipoprotein A-1 (ApoA-1). The first MSP, MSP1, was engineered with its sequence based on the sequence of A-1, but without the globular N-terminal domain of native A-1. The MSP1E3D1 variant of MSP1 differs from MSP1 in the following facets: (1) It deletes the first 11 amino acids in the Helix 1 portion of the original MSP1 sequence (which is known separately as MSP1D1). The MSP1D1 protein is an N-terminal histidine-tagged protein with a TEV protease cleavage site between the histidine-tag and the protein sequence. (2) It repeats the Helix 4 (H4), Helix 5 (H5) and Helix 6 (H6) sequences of the original MSP1 sequence between the parent Helix 6 (H6) and Helix 7 (H7) segments of MSP1D1.

Experimental Design and Results

Our ultimate goal is to successfully construct a well-functioning nanodisc, thus it is crucial to explore the construction process and identify suitable conditions for the nanodisc. Literature has already demonstrated that MSP1E3D1 can successfully construct nanodiscs, therefore, we have decided to use MSP1E3D1 as our target protein to explore the nanodisc construction process suitable for our experimental conditions.

We found the pMSP1E3D1 plasmid on Addgene, which can express MSP1E3D1 in E. coli. After transferring the pMSP1E3D1 plasmid into E. coli BL21 (DE3) cells, we obtained single colonies. PCR was performed on the single colonies, and the results were verified by agarose gel electrophoresis (Figure 1). Among the four selected single colonies, colony 4 most successfully obtained the target band.

Figure 1 four lanes from right to left: marker, colonies 1, 2, 3, and 4.

We proceeded with the expansion culture of the single colony. 0.2mM IPTG was added for induction, and the culture was incubated in a shaker at 16°C and 200 rpm for 16 hours. Verification was performed through SDS-PAGE (Figure 2). The two lanes on the far right are the added MSP1E3D1 samples, and the bands are very unclear. This suggests that the concentration of our samples may be too high.

Figure 2 The leftmost lane represents the protein molecular weight standard marker, while the two rightmost lanes show the crudely extracted MSP1E3D1 after IPTG induction.

The induced expression of MSP1E3D1 was unsuccessful, and we speculated that this might be caused by an excessively long expansion culture time or suboptimal induction conditions. Therefore, we decided to explore the optimal OD600 value for the expansion culture of MSP1E3D1 as well as the most suitable induction conditions.We conducted tests for scaling up the culture of single colonies under suitable cultivation conditions. The OD600 value was measured by taking bacterial liquid from the test bottles at regular intervals, and the relationship between OD600 value and cultivation time was obtained (Figure 3).

Figure 3 Relationship between OD600 value and cultivation time.

Based on the relationship between the obtained OD600 and cultivation time, we scaled up the culture of nine bottles of bacterial liquid, and added 0.2mM IPTG at nine different times for induced cultivation, which was verified by SDS-PAGE (Figure 4 a).Compared to the test lane (before induction), there is no significant change in the relative protein content in lanes 1-9, indicating that 0.2mM IPTG did not effectively induce the protein expression. However, from the electrophoresis results, it can be observed that lane 5 has a higher protein content, corresponding to an OD600 value of 0.605 before induction, suggesting that protein production is more efficient when the OD600 value of the bacterial culture reaches 0.6.Subsequently, we set up an IPTG gradient, added IPTG solutions of different concentrations to the bacterial liquid that reached the OD value for cultivation, and verified it through SDS-PAGE (Figure 4 b).A distinct MSP1E3D1 band is observed at an IPTG concentration of 0.8mM, suggesting successful induction. Therefore, an IPTG concentration of 0.8mM is selected.We need to further purify MSP1E3D1 to meet the requirements for the production of nanodiscs.

Figure 4(a) From left to right, the lanes are marker, test, 1-9.(b) From left to right,the lanes are: marker,test (without IPTG),0.1-0.9(IPTG concentrations).

After consulting literature and seeking advice from our supervisor, we have determined the purification method for MSP1E3D1. We resuspended the centrifuged precipitate using an alkaline buffer (with AEBSF and Triton added for protein stabilization), followed by sonication and recentrifugation. We employed Ni-NTA as the filler material and used four different washing buffers to remove impurities, then added elution buffer to obtain the target protein. The purification effect was verified through SDS-PAGE (Figure 5).It can be seen that in the lane of loading EB, the band of MSP1E3D1 is relatively obvious, proving that we have successfully obtained MSP1E3D1, but there are still many impurity proteins.

Figure 5 From left to right are marker, EB (target protein), WB4, WB3, WB2, WB1, supernatant passed through column, and supernatant not passed through column.

We decided to use AKTA for further purification of MSP1E3D1. We collected the effluent from six peaks of AKTA and verified it through SDS-PAGE (Figure 6a).A faint MSP1E3D1 band can be observed in Peak 1.Considering that the concentration of MSP1E3D1 might be low, we used an ultrafiltration tube to concentrate the effluent and then verified it again through SDS-PAGE (Figure 6b).This demonstrates the successful purification of MSP1E3D1.

Figure 6 (a) From left to right, the lanes are marker, Peak 1 to Peak 6.(b) From left to right, the lanes are marker, concentrated MSP1E3D1, and the liquid at the bottom of the ultrafiltration tube.

We have obtained a relatively pure MSP1E3D1, but its current concentration is still relatively low. In future experiments, we can continue to explore how to obtain MSP1E3D1 with higher concentration and purity.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 115
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 115
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 115
  Illegal BglII site found at 741
  Illegal XhoI site found at 637
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 115
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 115
  
• 1000
  COMPATIBLE WITH RFC[1000]