Protein constructs, expression, and purification

Full coding region of the Human NEU1 construct:

MKFLVNVALVFMVVYISYIYADRHHHHHHGSENDFGLVQPLVTMEQLLWVSGRQIGSVDTFRIPLITATPRGTLLAFAEARKMSSSDEGAKFIALRRSMDQGSTWSPTAFIVNDGDVPDGLNLGAVVSDVETGVVFLFYSLCAHKAGCQVASTMLVWSKDDGVSWSTPRNLSLDIGTEVFAPGPGSGIQKQREPRKGRLIVCGHGTLERDGVFCLLSDDHGASWRYGSGVSGIPYGQPKQENDFNPDECQPYELPDGSVVINARNQNNYHCHCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGAVVTSSGIVFFSNPAHPEFRVNLTLRWSFSNGTSWRKETVQLWPGPSGYSSLATLEGSMDGEEQAPQLYVLYEKGRNHYTESISVAKISVYGTL

Mouse NEU1:

MKFLVNVALVFMVVYISYIYADRHHHHHHGSEDDFSLVQPLVTMEQLLWVSGKQIGSVDTFRIPLITATPRGTLLAFAEARKKSASDEGAKFIAMRRSTDQGSTWSSTAFIVDDGEASDGLNLGAVVNDVDTGIVFLIYTLCAHKVNCQVASTMLVWSKDDGISWSPPRNLSVDIGTEMFAPGPGSGIQKQREPGKGRLIVCGHGTLERDGVFCLLSDDHGASWHYGTGVSGIPFGQPKHDHDFNPDECQPYELPDGSVIINARNQNNYHCRCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGALATSSGIVFFSNPAHPEFRVNLTLRWSFSNGTSWQKERVQVWPGPSGYSSLTALENSTDGKKQPPQLFVLYEKGLNRYTESISMVKISVYGTL

Human CTSA:

MKFLVNVALVFMVVYISYIYADRHHHHHHGSAPDQDEIQRLPGLAKQPSFRQYSGYLKGSGSKHLHYWFVESQKDPENSPVVLWLNGGPGCSSLDGLLTEHGPFLVQPDGVTLEYNPYSWNLIANVLYLESPAGVGFSYSDDKFYATNDTEVAQSNFEALQDFFRLFPEYKNNKLFLTGESYAGIYIPTLAVLVMQDPSMNLQGLAVGNGLSSYEQNDNSLVYFAYYHGLLGNRLWSSLQTHCCSQNKCNFYDNKDLECVTNLQEVARIVGNSGLNIYNLYAPCAGGVPSHFRYEKDTVVVQDLGNIFTRLPLKRMWHQALLRSGDKVRMDPPCTNTTAASTYLNNPYVRKALNIPEQLPQWDMCNFLVNLQYRRLYRSMNSQYLKLLSSQKYQILLYNGDVDMACNFMGDEWFVDSLNQKMEVQRRPWLVKYGDSGEQIAGFVKEFSHIAFLTIKGAGHMVPTDKPLAAFTMFSRFLNKQPY

Mouse CTSA with C32A surface mutation to possibly improve protein production:

MKFLVNVALVFMVVYISYIYADRHHHHHHGSAPDQDEIDALPGLAKQPSFRQYSGYLRASDSKHFHYWFVESQNDPKNSPVVLWLNGGPGCSSLDGLLTEHGPFLIQPDGVTLEYNPYAWNLIANVLYIESPAGVGFSYSDDKMYVTNDTEVAENNYEALKDFFRLFPEYKDNKLFLTGESYAGIYIPTLAVLVMQDPSMNLQGLAVGNGLASYEQNDNSLVYFAYYHGLLGNRLWTSLQTHCCAQNKCNFYDNKDPECVNNLLEVSRIVGKSGLNIYNLYAPCAGGVPGRHRYEDTLVVQDFGNIFTRLPLKRRFPEALMRSGDKVRLDPPCTNTTAPSNYLNNPYVRKALHIPESLPRWDMCNFLVNLQYRRLYQSMNSQYLKLLSSQKYQILLYNGDVDMACNFMGDEWFVDSLNQKMEVQRRPWLVDYGESGEQVAGFVKECSHITFLTIKGAGHMVPTDKPRAAFTMFSRFLNKEPY

Baculovirus generation and cell culture

The proteins were cloned into a modified pFastBac1 vector downstream of the polyhedrin promoter. The plasmids were transformed into E. coli containing the DH10MultiBac bacmid as well as the transposase required for integration of the gene into the bacmid (Geneva Biotech). Positive (white) colonies were grown in LB medium and bacmids were isolated. Bacmids were transfected into monolayer Sf9 cells using Cellfectin II (Gibco). Sf9 cells were grown at 27C in I-MAX medium supplemented with penicillin and streptomycin (Wisent BioProducts). After 4 days, the culture supernatant was added to a suspension culture of Sf9 cells and the baculovirus was further amplified for 3 days. The culture supernatant was filtered and the virus stock was added at 3.5% to a suspension culture of Sf9 cells for protein expression for 3 days.

Protein purification

The Sf9 expression culture was centrifuged and directly incubated with 20mL/L of HisPur Ni-NTA beads (ThermoFisher Scientific) for 2 hours with shaking. Beads were washed with buffer (25mM Tris-HCl pH7.5, 500mM NaCl, 10mM imidazole) and eluted with buffer containing 250mM imidazole. Proteins were concentrated and purified by size-exclusion chromatography with a Superdex 200 Increase column (Cytiva Life Sciences) in buffer (10mM Tris-HCl pH7.5, 100mM NaCl). NEU1 eluted as a very broad peak whereas CTSA eluted as a dimer. NEU1 was optionally further purified by anion exchange chromatography with a MonoQ column (Cytiva Life Sciences) with a buffer gradient (10mM Tris-HCl pH7.5, 100mM to 300mM NaCl) and concentrated to 10mg/mL. CTSA after size-exclusion was supplemented with 10mM Na-acetate pH4.5 and concentrated to 20mg/mL. Typical final yields were 5-10mg/L culture of NEU1 and 10-20mg/L culture of CTSA.