Sortase A conjugation of HIV-1 envelope trimers to ferritin nanoparticles

SOSIP Env conjugation to ferritin SOP

1    Specimens, Reagents, Materials and Equipment

1.1 Specimens

1.1.1    SOSIP-C-SORTA trimers

1.1.2    Ferritin

1.1.3    Sortase A

1.2 Reagents 

1.2.1    1M Tris, pH 7.4 (Sigma, T2319-1L)

1.2.2    5M NaCI (Santa Cruz Biotechnology, Inc sc-295833)

1.2.3    Calcium chloride dehydrate CaCl₂.2H₂O (Sigma C7902-500g) 

1.3 Materials

1.3.1    Diamond Grip Latex Gloves, Microflex-Large (Fisher Scientific, 11-462-67D)

1.3.2    Diamond Grip Latex Gloves, Microflex-Medium (Fisher Scientific, 11-462-67C)

1.3.3    Diamond Grip Latex Gloves, Microflex-Small (Fisher Scientific, 11-462-67B)

1.3.4    50 mL Conical tubes (VWR, 21008-940)

1.3.5    Kimwipes (VWR, 21905-049)

1.3.6    2 mL tubes (Sarstedt 72.694.005)

1.3.7    Ice

1.3.8    Ice Buckets 

1.3.9    Dry ice

1.3.10  250 mL storage bottle (VWR, 28199-758)

1.3.11  500 mL storage bottle (VWR, 28199-760)

1.3.12  25 mL BD Falcon Disposable Serological Pipets, Polystyrene, Sterile, Plugged, Individually Wrapped (Fisher Scientific, 13-668-2)

1.3.13  500 mL Corning filter (VWR, 28199-788)

1.3.14  1000 mL Corning filter (VWR, 28199-812)

1.3.15  Vivaspin 20, 10kDa (VWR, 95056-128)

1.3.16  RT-UNV-A-1000uL tips (Rainin 30389166)

1.3.17  RT-UNV-A-200uL tips (Rainin 30389190)

1.3.18  RT-UNV-A-20uL tips (Rainin 30389189)

1.4 Equipment 

1.4.1    Pipet Aid

1.4.2    Pipettors

1.4.3    Sorvall Legend RT Centrifuge

1.4.4    Hood

1.4.5    Nanodrop

1.4.6    Plate Reader

1.4.7    Scale 

1.4.8    Freezer

1.4.9    Fridge

1.4.10  FPLC

2   Protocol

2.1 Buffer Preparation 

2.1.1    1M CaCI₂: Weigh 14.7g of Calcium chloride dehydrate CaCl₂.2H₂O and add it to a 250ml bottle. Bring up volume to 100mL with Mini Q H₂O 

2.1.2    Conjugation Buffer: Prepare conjugation buffer in a a 500ml bottle and add 25ml of 1M Tris, pH 7.4, 15 ml of 5 M NaCI, and 2.5 ml of 1 M CaCI₂. Bring the volume up to 500 ml with MilliQ H₂O. Filter using sterile filter (0.22 μm) and store at 4° C.

2.2 Calculations

2.2.1    Make the calculations for the conjugation reaction with this website: https://www.physiologyweb.com/calculators/molar_solution_concentration_calculator.html

2.2.1.1 Calculate the reaction volume by imputing the protein's molecular weight, the mass of solute (mg), and the concentration (um). Click calculate to find the total solution volume (ul).

2.2.1.2 Calculate the amount of Ferritin by inputting molecular weight, total solution volume from the previous step (ul), and concentration (um).  Click Calculate to find the mass of solute (mg).

2.2.1.3 Calculate the amount of Sortase A by inputting molecular weight, total solution volume from step 2.2.1.1 (ul), and concentration (um).  Click Calculate to find mass of solute (mg).

2.3 Conjugation Procedure

2.3.1    Pipet 20 mL of cold conjugation buffer into a 50 ml conical tube. Place it on ice

2.3.2    Thaw SOSIP Protein: Add 1 mL of the cold conjugation buffer to each frozen aliquot SOSIP Protein. Leave on ice for 1 hour

2.3.3    Transfer SOSIP Protein into 10kDa Vivaspin 20

2.3.4    Rinse SOSIP Protein tubes with the rest of the conjugation buffer and transfer into 10kDa Vivaspin 20

2.3.5    Add the amount of the Ferritin from step 2.2.1.2 (mg) into the 10kDa Vivaspin 20

2.3.6    Add the amount of the Sortase A from step 2.2.1.3 (mg) into the 10kDa Vivaspin 20

2.3.7    Centrifuge 3000xg for 1 hour. Concentrate down until ~ 250 ul.

2.3.8    Transfer concentrated mix into a 1.5 mL tube.

2.3.9    Rinse the 10kDa Vivaspin 20 reservoir with conjugation buffer and transfer it into a 1.5 mL tube. Bring the volume up to 450ul 

2.3.10  Shake the 1.5ml tube gently overnight (16 hours).

2.3.11  After 16 hour incubation, place the reaction tube on ice.

2.3.12  Bring the volume of the reaction tube up to 530 uL to run size exclusion

2.3.13  Run size exclusion using the method “z-Superose 6 16 70 peak fractionation 0.5 ml inj 0.5 ml frac”

2.3.14  Select fractions symmetrically from peak 1 and peak 2 

2.3.15  Run Bradford Assay find concentration of your nanoparticle

2.3.16  Follow the SOSIP snap freezing protocol to make and freeze the nanoparticle aliquots. QC: 2 x 20 µg for EM; 20 µl for Endotoxin; 100 µg for BLI; and 40 µg for FPLC QC and 40ug for Gel