Protocol Annotation

1. Expression
1.1 Expression system selection E. Coli strain M15 [pREP4] is chosen for the most optimal
Recombinant plasmids veri ed in previous steps to contain the expression system because it works with the T5 promoter
gene of interest are transformed into the E. Coli strain M15 transcription-translation system and contains a high copy of
[pREP4] for the expression of the target protein. lacq mutation, enabling an ef cient and highly regulated
expression of the heterologous protein. In addition, it also has
the advantages of easy operation and low price.
1.2 Conditions for cell culture and induction In general conditions, the lac operon inhibits the expression of
(1) The successfully transformed E. coli is inoculated into the the target protein, and an inducer is needed to initiate the
LB medium containing 100 μg/mL ampicillin and 25 μg/mL expression. The reason for using IPTG as an inducer instead of
kanamycin and cultured overnight at 37°C on a shaker. 1 lactose is that it can recognise the repressor but can’t be broken
Cells containing mL sample is kept and stored at -20°C at this step for down by the cells, maintaining the intracellular concentration of
the target protein subsequent SDS-PAGE analysis. the inducer and thus maintaining a high expression of the target
(2) When the OD600 of cell culture reaches 0.5~0.7, lactose protein.
analogue isopropyl-β-D-thiogalactoside (IPTG) at a nal
concentration of 1mM is added for the induction of
expression of the target protein. Keep culturing for an
additional 4~5 hours at the same condition after the
induction. 1 mL sample is kept and stored at -20°C at this
step for subsequent SDS-PAGE analysis.
2.1 Cell lysis and crude extraction Cell lysis and crude extraction are crucial to the yield of the
2. Extraction (1) Centrifuge the induced cell culture at 4000× g for 20 target protein. Protein losses can be reduced by optimising cell
minutes to collect the cell pellet. lysis conditions to increase the release of target proteins from
(2) Resuspend the cell pellet with 5mL lysis buffer and add the expression host, optimising homogenisation methods to
lysozyme to a nal concentration of 1 mg/mL. Incubate on separate target proteins from cell debris, adding DNAase/
ice for 30 minutes. RNAase to reduce the viscosity of the cell lysate and improving
(3) Sonicate the cell lysate at 200~300W for 10 seconds with experimental conditions to reduce the denaturation of target
an interval of 10 seconds and repeat 6 times. proteins.
(4) Centrifuge the cell lysate at 10,000× g for 20~30 minutes By querying the UniProt database, it is found that the target
at 4°C, and the supernatant is the crude extraction protein has good solubility, so the crude extract target protein is
solution of the target protein. Keep the sample on ice. 1 present in the supernatant.
mL sample is kept and stored at -20°C at this step for
Crude extract of subsequent SDS-PAGE analysis.
the target protein 2.2 SDS-PAGE justi cation By querying the UniProt database, the size of the target protein
(1) Mix the previously kept un-induced, induced, and crude was found to be 51442 Da. Therefore, the expected results of
extraction samples with an equal amount of 2× SDS-PAGE SDS-PAGE are: the un-induced sample has no evident band
loading buffer. near 51kDa, while the induced sample and crude extract
(2) The above samples were incubated at 95°C for 5 minutes sample have evident bands at around 51kDa. This can justify
and then centrifuged at 15,000× g for 1 minute. the success of induction and crude extraction of target proteins.
(3) Load 20μL of each sample on a 12% SDS-PAGE gel.
Reserve one lane and add 10 μL of standard protein. Run
the gel according to standard protocol and image the gel to
justify the success of induction and crude extraction.

3. Puri cation 3.1 Fast protein liquid chromatography (FPLC) puri cation
(1) Pour the fully resuspended 50% Ni-NTA super ow slurry
Since the puri ed target protein needs to be assayed for
enzyme activity subsequently, a puri cation protocol that allows
slowly into the column to avoid bubbles. Based on the protein under native conditions is required. Ni-NTA Super- ow
protein content in the crude extract, estimate the required column is chosen because it works with 6× His tag attached to
slurry volume at a ratio of 5~10 mg of protein per mL. Wait the N-terminal of the target protein for highly ef cient
for the resin to settle, assemble the column with the puri cation.
system, and determine the volume of resin as the column The purpose of equilibration is to settle the resin into a
volume (CV). homogenised state, ensuring a stable and ef cient puri cation.
(2) Equilibrate the column with 5 CV of cell lysis buffer at a ow The purpose of washing is to elute contaminant proteins that
rate below 2 mL/min. Collect samples for every 1 mL of the have low af nity to the column matrix to achieve puri cation.
owthrough for spectrophotometer analysis subsequently. The purpose of elution is to detach the puri ed protein from the
Samples are labelled as A1, A2, A3, etc. column matrix and collect it in the owthrough for the
(3) Load the target protein crude extract into the column and subsequent analysis.
wash with 5~10 CV of lysis buffer. Collect samples for
every 1 mL of the owthrough for spectrophotometer
analysis subsequently. Samples are labelled as W1, W2,
W3, etc.
Puri ed target
(4) Elute the target protein with ~5 CV of elution buffer, and the
protein with the owthrough is the puri ed target protein solution. Collect
tag on samples for every 1 mL of the owthrough for
spectrophotometer analysis subsequently. Samples are
labelled as E1, E2, E3, etc.
3.2 Spectrophotometer justi cation The predicted results of the elution curve are as follows:
(1) Set the wavelength of the spectrophotometer to 280 nm. In the rst few fractions of sample A, absorption peaks can be
Add 1 mL of lysis buffer to the cuvette and use this sample observed, and then the absorption stabilises to the baseline,
as blank. proving the success of column equilibration. In the rst few
(2) Add 1 mL of each fraction sample to the cuvette and fractions of sample W, evident absorption peaks can be
measure the absorbance of all samples. observed, and then the absorption stabilises to the baseline
(3) Plot the absorbance versus the order of the fractions into gradually, which proves the success of the removal of
an elution curve graphic, examining and justifying the contaminant proteins. In the rst few fractions of sample E,
success of the puri cation of the target protein. evident absorption peaks can be observed, and then the
absorption quickly drops to the baseline, which proves the
successful elution of the target protein.
4.1 Cleavage Factor Xa protease is tailored for the cleavage of 6× His tag
4. Tag removal (1) Mix the puri ed target protein solution and an equal volume produced by the pQE-30 Xa vector. It recognises the amino acid
of 1× reaction buffer and equally divide it into 4 portions. sequence Ile-Glu-Gly-Arg and conducts the digestion, removing
Dilute factor Xa protease with 1× reaction buffer to three the tag from the target protein.
serial dilutions with concentrations of 1.0, 0.2 and 0.05 The condition of the reaction buffer contains 20 mM Tris·Cl, pH
units per μL. 6.5; 50 mM NaCl; 1 mM CaCl2 .
(2) Add 1 μL of each factor Xa protease dilution to 3 puri ed
target protein samples respectively, and adjust all 4
samples with 1× reaction buffer to a nal volume of 40 μL.
(3) After incubation at room temperature for 3, 6, 9, and 16
hours, take 8 μL aliquots for each incubation time from
each of the four samples and mix them with 2 μL of 5×
SDS-PAGE loading buffer. SDS-PAGE analysis is used to
determine protease cleavage ef ciency, and the sample
with the highest ef ciency is decided as the optimal
condition for tag cleavage. Scale up for the subsequent
cleanup.
Pure, intact and
native Neuraminidase 4.2 Cleanup of the digested protein After the cleavage with factor Xa protease, the target protein
for enzymatic assay (1) Thoroughly resuspend the Xa protease removal resin and solution can be further puri ed by removing the Xa protease by
add an appropriate amount of slurry to a centrifuge tube. af nity chromatography using Xa removal resin and removing
Add 10 resin-volume of 1x reaction buffer, fully resuspend the 6× His tag by Ni-NTA agarose.
by inversion. Centrifuge at 1000× g for 5 minutes and
discard the supernatant. Repeat twice.
(2) Add the cleavage reaction solution to the equilibrated
resin, mix gently and incubate at room temperature with
shaking for 10 minutes. Centrifuge at 1000× g for 5
minutes, and the supernatant is the target protein solution
from which Xa protease is removed. Adjust the pH of the
solution to 7.5 for subsequent puri cation.
(3) Thoroughly resuspend the Ni-NTA agarose and add an
appropriate amount of slurry to a centrifuge tube.
Centrifuge at 1000× g for 1 minute and discard the
supernatant.
(4) Add the cleavage reaction solution to the equilibrated
resin, mix gently and incubate at room temperature with
shaking for 10 minutes. Centrifuge at 1000× g for 1
minute, and the supernatant is the target protein solution
from which Xa protease and 6× His tag is removed.
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Enzymatic Assay

Protocol Annotation

1. A continuous spectrophotometric detection method is chosen for the

enzymatic assays of neuraminidase based on the different properties of the
substrate 2-O-(p-nitrophenyl)-N-acetylneuraminic acid (pNP-Neu5Ac) and the
product p-nitrophenol (pNP), using a Cary 100 UV-Vis spectrophotometer.

2. The concentration of substrate pNP-Neu5Ac ranges from 200 μM to 400 μM,

and the initial velocity of the reaction is measured under different enzyme NA
MES buffer contains 2-(N-morpholino)ethanesulfonic acid, and the pH is
concentrations (14~140 nM) at a xed wavelength of 400nm and 30℃. The
adjusted to 6.0. This experiment is conducted at 30°C.
buffer for all enzymatic assays is 20 mM MES buffer, pH 6.0. Make 3 duplicates
per group.

3. Plot the initial velocity versus substrate concentration and integrate the data Enzyme activity is re ected by changes in the absorption peak of the reaction
into a Michaelis-Menten curve to determine the Vmax and calculate various system, in which a UV-visible spectrophotometer is used. The absorption will
dynamic parameters. detected at 400nm, focusing on the accumulation of the product pNP.

Vmax can be read directly through the Michaelis-Menten curve, and Km is

calculated by substituting 1/2 Vmax into the Michaelis-Menten equation. The
4. The accumulation of pNP in the reaction system will bring an absorption
reaction with heat-inactivated NA is added as a blank control to determine the
peak at 400nm and red-shift the substrate absorption peak originally located at
velocity of the uncatalysed reaction. Compare Vmax and Km calculated with
300nm to 315nm.
data in the literature (Vmax=0.28 μM/min, Km=34 μM) to determine if the assay
truly conforms to Michaelis-Menten kinetics.
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