Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations.

Kulakova A, Indrakumar S, Sønderby Tuelung P, Mahapatra S, Streicher WW, Peters GHJ, Harris P, Sci Rep 10(1):10089 (2020) Europe PMC

SASDHX8 – Human serum albumin/Neprilysin fusion protein (HSA-NEP): 10 mM histidine, pH 6.5

Neprilysin - G400V mutant
Human serum albumin - C58S mutant
MWexperimental 147 kDa
MWexpected 147 kDa
VPorod 258 nm3
log I(s) 1.27×10-1 1.27×10-2 1.27×10-3 1.27×10-4
Neprilysin - G400V mutant Human serum albumin - C58S mutant small angle scattering data  s, nm-1
ln I(s)
Neprilysin - G400V mutant Human serum albumin - C58S mutant Guinier plot ln 1.27×10-1 Rg: 4.6 nm 0 (4.6 nm)-2 s2
(sRg)2I(s)/I(0)
Neprilysin - G400V mutant Human serum albumin - C58S mutant Kratky plot 1.104 0 √3 sRg
p(r)
Neprilysin - G400V mutant Human serum albumin - C58S mutant pair distance distribution function Rg: 5.1 nm 0 Dmax: 16 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Neprilysin - G400V mutant Human serum albumin - C58S mutant EOM/RANCH model
Neprilysin - G400V mutant Human serum albumin - C58S mutant EOM/RANCH model
Neprilysin - G400V mutant Human serum albumin - C58S mutant EOM/RANCH model
Neprilysin - G400V mutant Human serum albumin - C58S mutant EOM/RANCH model
Neprilysin - G400V mutant Human serum albumin - C58S mutant EOM/RANCH model
Synchrotron SAXS data from solutions of the HSA-NEP fusion in 10 mM histidine, pH 6.5 were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). 20 successive 0.050 second frames were collected at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentration (concentration UNKNOWN) were merged with the highest concentration high angle data (concentration UNKNOWN) to yield the final composite scattering curve.

The engineered protein, expressed in Chinese hamster ovary (CHO) cells, is a fusion between HSA and human Neprilysin connected by a GGGGS amino acid linker. CAUTION! The effect of severe repulsive interparticle interference are evident in the Guinier region that has been entirely removed for EOM modelling calculations.

Neprilysin - G400V mutant (NEP(G400V))
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   80.3 kDa
 
UniProt   P08473 (52-750)
Sequence   FASTA
 
Human serum albumin - C58S mutant (HSA(C58S))
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   66.5 kDa
 
UniProt   P02768 (25-609)
Sequence   FASTA