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DENV NS2B-NS3 plasmids don't specify which DENV Serotype #58

Open jchodera opened 1 year ago

jchodera commented 1 year ago

We don't currently specify which serotype our DENV Serotype NS2B-NS3 plasmids come from: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-DENV-NS2B-NS3

Also, the constructs say things like:

Tags and additions: N-terminal, TEV protease cleavable hexahistidine

but then have a construct sequence that lacks both the N-terminal HHHHHH and a typical ENLYFQ|S(G,A) TEV cleavage sequence:

Construct protein sequence: NS2B3: SMADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLGGGGSGGGGAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK

cc @LizbeK

jchodera commented 1 year ago

Submitting this sequence to blastp shows that it seems to be closest to (but not identical to) DENV-2?

233: ref|NP_056776.2| polyprotein [Dengue virus type 2] ADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK

jchodera commented 1 year ago

It does appear to be identical to ref|NP_056776.2| polyprotein [Dengue virus type 2] except for a piece swapped out with a soluble linker:

SMADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTL............GGGGSGGGG..............AGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK
..ADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK

Shouldn't our sequences list the entire translated sequence, rather than just the post-TEV cleavage product?

jchodera commented 1 year ago

From Mike Fairhead:

Karla's reference sequence Dengue virus type 2 (strain 16681) polyprotein mRNA, complete cds GenBank: U87411.1 NS2B from polyprotein LNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQR NS3 protease from polyprotein LEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQT Uniprot: P14340 (closest match) https://www.uniprot.org/uniprotkb/P14340/entry BRENDA E.C.C. 3.4.21.91 https://www.brenda-enzymes.org/enzyme.php?ecno=3.4.21.91 Scarab sequences Crystallization construct(s) No current construct Assay construct(s) NS2B-NS3 Fusion proteins for enzymatic assays (based on Karlas reference sequence) QQ01D2VNS2B-c001 MHHHHHHSSGASWSHPQFEKGGGSGGGSGGSAWSHPQFEKGSGVDLGTENLYFQ//SMADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLGGGGSGGGGAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK