Optimization of the Production of Covalently Circularized Nanodiscs and Their Characterization in Physiological Conditions

Abstract

Lipid nanodiscs are widely used platforms for studying membrane proteins in a near-native environment. Lipid nanodiscs made with membrane scaffold proteins (MSPs) in the linear form have been well studied. Recently, a new kind of nanodisc made with MSPs in the circular form, referred to as covalently circularized nanodiscs (cNDs), has been reported to have some possible advantages in various applications. Given the potential of nanodisc technology, researchers in the field are very interested in learning more about this new kind of nanodisc, such as its reproducibility, production yield, and the possible pros and cons of using it. However, research on these issues is lacking. Here, we report a new study on nanodiscs made with circular MSPs, which are produced from a method different from the previously reported method. We show that our novel production method, detergent-assisted sortase-mediated ligation, can effectively avoid high-molecular-weight byproducts and also significantly improve the yield of the target proteins up to around 80% for larger circular MSP constructs. In terms of the application of circular MSPs, we demonstrate that they can be used to assemble nanodiscs using both synthetic lipids and native lipid extract as the source of lipids. We also show that bacteriorhodopsin can be successfully incorporated into this new kind of cND. Moreover, we found that cNDs have improved stability against both heat and high-concentration-induced aggregations, making them more beneficial for related applications.

A Split‐Intein‐Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins

“…Furthermore, the long‐term stability of nanodiscs is also essential for biochemical assays, for example, for probing amyloid binding to lipid surfaces . To improve the homogeneity of nanodiscs for structural applications, circular MSP (cMSP) proteins have been produced by using sortase A mediated protein ligation, However, this additional enzymatic reaction needs to be optimized for each batch of enzyme, and ligation yields can vary considerably.…”

“…The mechanism of intein‐mediated ligation has been described by Kent and co‐workers for the semisynthetic preparation of protein–peptide adducts by using proteins containing an N‐terminal cysteine and peptides harboring C‐terminal thioesters; this method was named expressed protein ligation (EPL) . A major advantage of the intein ligation method is the direct production of circular proteins in vivo without the need for a downstream enzymatic reaction for MSP circularization, as required with sortase A mediated ligation; this reduces the time for the recombinant production and purification of cMSPs from >4 to 2 days. Furthermore, we obtain consistently high yields of cMSPs (Table ).…”

“…A small portion of the cMSP tends to covalently dimerize in vivo to give rise to a band at about 35 kDa (cMdi). By using sortase A, oligomerization of the MSP is a prominent side reaction that can be reduced by lowering the MSP concentration and/or the use of detergents …”

“…Using this strategy, we inserted a large variety of MSP constructs into our split‐intein plasmid. For the very small MSP variants MSPΔ H 45 and MSPΔ H 4‐6 or the large MSP26 variant shown to form 15 nm nanodiscs because it most likely adopts a double ring around a lipid patch, we removed the His 6 ‐tags flanking the split inteins and added the His 6 ‐tag to the final cMSP. Thus, Ni‐NTA and Q‐sepharose chromatography in 8 m urea could be used as a main purification strategy (see the Methods section in the Supporting Information).…”

Beyond detergent micelles: The advantages and applications of non-micellar and lipid-based membrane mimetics for solution-state NMR

“…In order to achieve covalent circularization, a Cterminal SortaseA recognition site (LPETG) was fused to the MSP to be ligated to an N-terminal Glycine residue of the MSP using SortaseA enzyme [73,74]. Intramolecular circularization can be facilitated by using diluted MSP solutions of around 10 µM concentration and by adding mild detergents, such as Triton-X100 [75]. Furthermore in another study, the properties of the MSP were engineered to optimize solubility, an approach that further enhanced the yields of circular MSP after ligation with SortaseA [76].…”