Product Type: ELISA Kit
Storage: All kit reagents 2°C to 8°C
Target expression system: P. pastoris
Species Group: Yeast
Species: P. pastoris
Assay format: 96-well plate
Time to result: ~1 hr. 50 minutes or ~3 hours. 50 minutes
LOD: <0.3ng/mL or <1ng/mL
LLOQ: ~0.4ng/mL or ~1.5ng/mL
Recommended thinner: I028
Resume
Leishmania Donovan is the main cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality, second only to malaria. There is currently no human vaccine available. A 36-kDa L. donovani nucleoside hydrolase surface protein (LdNH36) was previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris HCP Elisa Kit and its purification at 20 L scale to establish suitability for future pilot scale manufacturing. To improve process development efficiency and ensure reproducibility, all 4 N-linked glycosylation sites that were shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues.
The LdNH36 mutant (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modelling, and the mutated amino acids are located outside the main immunogenic domain. The immunogenic properties of the recombinant protein LdNH36-dg2 were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)).
The mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive with LdNH36-dg2 and wild-type LdNH36. While point mutations affected the hydrolase activity of the enzyme, IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of wild-type LdNH36. The results indicate that LdNH36-dg2, as expressed and purified from P. pastoris, is suitable for further scale-up, manufacturing, and testing in support of future early phase clinical trials in humans.
Keywords: CpG oligodeoxynucleotide, fermentation, Leishmania donovani, poly(lactic-co-glycolic) acid (PLGA), purification
Methods
- Detection of host cell proteins (HCP)
The presence of host cell proteins was assessed by transferring the proteins from a 4–12% Bis-Tris SDS-PAGE gel (performed as described above) to a nitrocellulose membrane and probing with a 1:1000 dilution of an anti -P of goat. pastoris primary (Cygnus Technologies; Southport, NC) followed by a 1:5,000 dilution of a rabbit anti-goat alkaline phosphatase (KPL)-labelled secondary antibody. BCIP-NBT (KPL) was used for colourimetric detection.
- Enzymatic digestion of glycans
To assess the presence of N-linked glycosylation of the recombinant protein LdNH36-dg2, a PNGase F assay (New England Biolabs; Ipswitch, MA) was performed according to the manufacturer’s instructions. Digestion was analyzed with a 4-20% reduced Tris-glycine gel (Life Technologies) transferred to a nitrocellulose membrane. An anti-LdNH36 colourimetric Western blot was performed as described above.
- High-performance liquid chromatography
A Prominence flash liquid chromatography (UFLC) system from Shimadzu (Kyoto, Japan) with a photodiode array (PDA) detector and Phenomenex (Torrance, CA) tandem columns (Yarra 3µ SEC-2000 followed by Yarra 3µ SEC) was used. -3000). Injections of 50 µl were done at a flow rate of 0.5 ml/min in 30 mM Tris pH 7.5, and 150 mM NaCl at room temperature. Data analysis was performed on the 280 nm extracted chromatograph. To determine the molecular weight of the peak, a Bio-rad gel filtration standard (Hercules, CA) was injected to create a curve of distribution coefficient (Kd) versus the log of MW.
- Dynamic Light Scattering (DLS)
Dynamic light scattering was performed with a Wyatt (Santa Barbara, CA) DynaPro 384-well plate reader at room temperature in SEC200 elution buffer (30 mM Tris pH 7.5, 150 mM NaCl). Data were obtained by averaging 10 measurements, which were collected at 5-s intervals. Dynamic v7 software was used to estimate the hydrodynamic radius from a cumulative fit of the DLS autocorrelation function, assuming a globular protein.
- Structural modelling
The structure of LdNH36-dg2 was predicted through comparative modelling, using a semi-automated Swiss Model and all visualizations/figures were generated with PyMOL (The PyMOL Molecular Graphics System, Schrodinger, LLC). The published structure of a homologue, the L. major nucleoside hydrolase, was used as a template PDBsum was used to determine the amino acids present at the tetramer interface.
