Yishun Junior College_Lou Wei Hao Darren_ SSEF report_BC020

Lou Wei Hao Darren (BC020)

1

1. Background and Purpose of Research Area

Dengue virus (DENV), a viral disease transmitted via arthropods, in particular, Aedes agypti mosquito.

DENV belongs to the Flavivirigenus family, genus Falvivirus. Besides the four serotypes of DENV,

other viruses that come under the same virus family of Flavivirus include the Japanese encephalitis

virus, tick-borne encephalitis virus, West Nile virus and yellow fever virus [1]. DENV has become

increasingly prevalent worldwide where a recent study estimates that 390 million people are infected

each year with DENV, with 96 million infections exhibiting disease symptoms per annum [2]. With

increasing urbanisation and the continued trend of global warming, cases of DENV would only rise,

placing more than 2.5 billion people are at risk of contracting DENV [3]. In addition, it is estimated that

the cost of DENV in the Western Hemisphere alone amounts to $2.1 billion per year [2]. Till today, a

successful vaccine, chemotherapeutic agent or inhibitors that target DENV has yet to be discovered.

Thus, there is a critical need to create a vaccine that targets DENV [4-5].

DENV is an enveloped virus of approximately 50 nm, containing a 11 kb positive sense single strand

RNA genome (ssRNA)[6]. The genome is made up of a 5’ un-translated region (UTR) with a type I cap

(m7GppAm) a single long open-reading frame and a 3’ UTR [7]. After translation, a long polypeptide

chain containing ten proteins encoded by the open reading frame is formed. These ten proteins consist

of three structural proteins, namely the capsid (C), pre-membrane (prM) and the envelope (E), as well as

seven nonstructural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Figure 1) [8-9].

Each of these nonstructural proteins plays a part in viral replication and immune evasive functions. Figure 1 Schematic representation of DENV membrane proteins. NS4B is a membrane protein with three trans-membrane regions while NS3 has a looped domain. C, PrM and E represent caspid, pre-membrane and envelope respectively [10].

The primary function of NS4B known is inhibition of interferon (IFN) alpha/beta. In particular, it was

observed that the expression of NS4B caused IFN-induced signal transduction cascade to be blocked by

interfering with STAT1 phosphorylation [11]. IFN plays a crucial role in defending a host from any

viral infections, which has been seen in the case of mice deficient in the IFN pathway [12-13]. This in

turn results in the pathogenicity of DENV, making it crucial for the viral replication process [14-15].

NS3 (618 amino acids) is a multifunctional protein that has a serine protease (NS3pro) domain at its

N-terminus, while the C-terminus contains an ATP-driven helicase (NS3 helicase). RNA triphosphate is

found at its C-terminal end [16-17]. The NS3pro (NS2B being a cofactor) helps to carry out proteolytic

Endoplasmic reticulum


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cleavage of the translated viral polyprotein at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5

junctions [18-20]. NS3 helicase has a molecular weight of 75 kDa [2]. It helps in unwinding of the

RNA secondary structure in the 3’ UTR, which in turn helps to start the synthesis of the negative sense

single stranded RNA (ssRNA). The negative sense ssRNA is subsequently used as a template for the

production of additional positive sense ssRNA [21]. It also helps to unwind the RNA duplexes between

the positive and negative sense ssRNA during viral replication [22-23]. It is important to note that the

unwinding activities of NS3 helicase have been demonstrated to be essential in DENV replication

through site-directed mutagenesis experiments, making it a target in the design of inhibitors,

chemotherapeutic agents or vaccines [24-26].

Little is known about the interactions between NS3 and NS4B. However, recent studies have shown that

the interactions between NS4B and NS3 caused NS3 to dissociate from the ssRNA and promote

unwinding of duplexes in the 3’ UTR. These duplexes form during the viral replication would promote

its unwinding abilities and thus aid in the replication process [6]. In particular, a report has shown that

the loop (molecular weight of 14.0 kDa) between trans-membrane segments 1 and 2 is important for

interactions with the helicase domain of NS3 [27]. Thus, the interaction between these two proteins

provides a new drug target for inhibiting the function of NS3 helicase, leading to the inhibition of

DENV replication. The objective of the project is to successfully express and purify both D2 NS4B loop

and NS3 helicase through immobilised metal ion affinity chromatography (IMAC) and gel filtration

chromatography for future binding studies like nuclear magnetic resonance (NMR) titration and

isothermal titration calorimetry (ITC).

2. Materials and methods

2.1 Expression of D2 NS3 helicase

Transformed BL21DE3 cells containing plasmid pET32, containing thiodoxin tag, histidine tags (his-

tag) and enterokinase active site sequences, and D2 NS3 helicase gene were inoculated in 100 ml of

unlabeled M9 minimal medium containing ampicillin (1:1000 dilution), which was then incubated at

37°C, 200 rpm overnight. BL21 DE3 cells were chosen for expression purposes as past research have

showed that it had the highest level of protein expression among C41, C43 and Rosetta DE3 cells [6].

The inoculation was transferred to a flask with 900 ml of unlabeled M9 medium containing ampicillin.

Initial optical density (O.D) of the culture was taken. The medium was then incubated at 37°C, 200 rpm.

Induction of protein with 1M of isopropyl 1-thio-beta-D-galactopyranoside (1:1000 dilution) was

carried out when an O.D at 600 nm of 0.6-0.8 was reached. The culture was then incubated at 25 °C,

200 rpm and left overnight. The overnight culture then underwent centrifugation at 8000 rpm for 15

minutes in which the desired cells were collected as pellets. The pellets were re-suspended using 40 ml

of re-suspension buffer [20 mM of NaPO4 (pH 7.2) and 0.5 mM NaCl].


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2.2 Purification of D2 NS3 Helicase

2.2.1 Immobilised metal ion affinity chromatography (IMAC)

Re-suspended pellets underwent sonication (15 min process time; 5 s between intervals; 40 nm

amplitude) while kept in an ice bath. Cell lysates were cleared by centrifugation at 20,000 rpm for

30 minutes. NS3 helicase was purified by IMAC using Ni2+ nitrilotriacetic acid (NTA) beads, which had

already been pre-equilibrated in a gravity column. The supernatant containing his-tagged NS3 helicase

proteins was then added into it. The proteins were incubated at 4°C for 1 hour with continuous rolling.

Proteins were then washed with 10 x column volume of washing buffer [20 mM NaPO4 (pH 7.2), 1 M

NaCl and 20 mM imidazole] and was eluted with 2 x column volume of elution buffer [20 mM NaPO4

(pH 6.0), 0.5 M NaCl, 0.5 M imidazole]. Flow through after loading and elution were collected.

Samples of cell lysates, supernatant (load), flow through and three elution fractions (E1, E2, E3) were

collected for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).

2.2.2 Gel Filtration Chromatography

Elution fractions that contained D2 NS3 helicase (75 kDa) were further concentrated to 5 ml, which was

then further purified by gel filtration chromatography with a gel filtration column (Superdex 16/60

200hr). Digestion buffer (pH 7.4) with 20mM of Tris-HCl (pH 7.8) and 150 mM of NaCl was used.

Fractions of 5 ml were collected during the process. Fractions containing protein were collected and

analysed using SDS-PAGE.

2.2.3 Digestion of Proteins

Digestion was carried out on the 5 ml fractions containing the protein, confirmed by SDS page gel

electrophoresis. 5 ml fractions containing NS3 helicase were digested with 5 ul of enterokinase along

with 2 mM of CaCl2. Samples of the protein undergoing digestion were taken at 0 hr, 1 hr, 2 hr, 4 hr and

16 hr after the addition of enterokinase for SDS page gel electrophoresis to confirm protein digestion.

Digested proteins underwent another round of gel filtration chromatography and verified by SDS-

PAGE. Selected fractions were combined and concentrated to 0.5 ml at 3600 rpm and 4°C.

2.2.4 Quantification of Protein

Protein absorbance was measured using a spectrophotometer. Protein concentration was calculated

using the Beer-Lambert Law, A = ebc, where e is extinction coefficient, b is the path length of the

sample and c is the concentration of the protein. Concentrated protein (0.5 ml) was flash frozen with

liquid nitrogen and stored at -80°C.

2.3 Expression and Purification of D2 NS4B Loop

Expression of D2 NS4B loop (amino acid number 121-169) in BL21 DE3 was carried out using the

same protocol. Rather than 1mM of IPTG, 0.5mM of IPTG was used to induce protein production.

Incubation was carried out at 18 °C overnight rather than 25 °C. 100ul of Tobacco Etch Virus Nuclear

Inclusion endopeptidase (TEV protease) was used in replacement of enterokinase.


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0 0.2 0.4 0.6 0.8 1

1.2 1.4 1.6

32.9

35.8

38.7

41.6

44.5

47.5

50.4

53.3

56.2

59.1

62

65

67.9

70.8

73.7

76.6

79.6

82.5

85.4

88.3

Absorbance/m

g ml-‐1

Volume/cm3

UV light (215nm)

UV light (280nm)

GF1

GF2

M Tota

l

Load

Elution

Digesti

on

15-

10-

50-

25-

20-

75-

250-

100-

150-‐

37-

(A) D2 NS3

he

licas

e (P

urified

and

conce

ntrated

)

NS3 helicase pre- digestion

NS3 helicase post-digestion

-‐0.05

0

0.05

0.1

0.15

0.2

0.25

32.5

35

37.5

40

42.5

45

47.5

50

52.5

55

57.5

60

62.5

65

67.5

70

72.5

75

77.5

80

82.5

85

87.5

90 Ab

sorabance/ mg ml-‐1

Volume/ cm3

UV light (215nm) UV light (280nm)

3. Results and Discussion

3.1 Protein Purification

Figure 2 (A) SDS PAGE of D2 NS3 helicase purification. M represents protein molecular marker; Total is the total cell lysate, load represents supernatant and elution is the fraction with the highest concentration purified by Ni-NTA resin; GF1 is purified protein after first round of gel filtration; Digestion contains enterokinase-digested proteins; GF2 is purified protein post-digestion; last lane on right contains final purified and concentrated D2 NS3 helicase. Figure 2 (B) Gel filtration chromatography of gel filtration-purified D2 NS3 helicase post-elution (GF 1). Figure 2 (C) Gel filtration chromatography of purified NS3 helicase, post-digestion (GF 2).

Bands on the gels indicate the presence of protein, with the band’s position indicating its molecular

weight. From Figure 2A, it was found that NS3 helicase was successfully purified as the elution band

corresponds to the molecular weight of NS3 helicase of 75.0 kDa pre-digestion. Fractions of GF1 and

GF2 were collected between 42 ml to 60 ml and 40 ml to 80 ml respectively (Figure 2B and 2C

respectively).

kDa

(B) (C) (B)


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Figure 3 (A) Purification of D2 NS4B loop. M represents protein molecular marker; Total is the total cell lysate, load represents supernatant and elution is the fraction with the highest concentration purified by Ni-NTA resin; GF1 is purified protein after first round of gel filtration; Digestion contains enterokinase-digested proteins; GF2 is purified protein post-digestion; last lane on right contains final purified and concentrated D2 NS4B loop.

Figure 3(B) Gel filtration chromatography of gel filtration-purified D2 NS4B loop post-elution (GF 1). Figure 3(C) Gel filtration chromatography of purified NS4B loop, post-digestion (GF 2).

From Figure 3A, as the elution band correspond with the molecular weight of D2 NS4B loop, it was

concluded that NS4B loop had been successfully purified. The eluate of GF1 and GF2 for NS4B loop

was collected from 65ml to 85 ml and 50ml to 118ml respectively ( Figure 3B and 3C respectively).

3.1.1 Dissociation activity of D2 NS4B loop

In the gel filtration chromatography of digested D2 NS4B loop, it was observed that there were two

peaks from between 60 ml-83 ml and 85 ml-120 ml (Figgure 3B) rather than just one peak from

between 60 ml-83 ml (Figure 3C). This suggested that NS4B loop had undergone dissociation from a

dimer to a monomer, which could be due to slight changes in pH and/or salt concentration. Many

proteins are active in when dimerised [28].Thus, by dissociating into a monomer, it is possible that the

functionality of NS4B loop would cease, hindering the viral replication process. Conditions that could

have led to the dissociation would be explored for anti-DENV drug development.

-‐0.5

0

0.5

1

1.5

30

33.8

37.5

41.3

45

48.8

52.5

56.3

60

63.8

67.5

71.3

75

78.8

82.5

86.3

Absorbance/ mg ml-‐1

Volume/ cm3


-‐0.2

0

0.2

0.4

0.6

0.8

1

53.7

57.4

61.1

64.8

68.5

72.2

75.9

79.6

83.3

87

90.7

94.4

98.1

101.8

105.5

109.2

112.9

116.6

Absorbance/ mg ml-‐1

Volume/ cm3


10-

50-

25-

15-

75-

250-

100-

150-

37-‐

GF1

GF2

M

Total

Load

Elution

Digesti

on

NS4B

Loop

(Purif

ied an

d

conce

ntrated

)

(A)

20-

NS4B loop pre- digestion NS4B

loop post- digestion

kDa

(B) (C)


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3.1.2 Yield of Proteins by IMAC

The presence of more protein bands other than NS3 helicase in Figure 2A as compared to NS4B loop in

Figure 3A implies that the purification of D2 NS3 helicase by IMAC resulted in the collection of more

junk proteins as compared to that of D2 NS4B loop. This suggested that the junk proteins produced

along with NS3 helicase could have a stronger affinity with the Ni2+ resin such that some of the junk

proteins might contain histidine tags/clusters that allow them to bind to the nickel resin as well.

Purification of NS3 helicase can be improved by using on-column digestion, where enterokinase, nickel

beads and the supernatant are first mixed. This allows the his-tag to be cleaved from the helicase, thus it

is not bound to the resin. At the same time, junk proteins with his-tag/clusters would remain bounded to

the resin. Thus, when the mixture is loaded back onto the column, flow through collected would contain

mostly NS3 helicase as the junk proteins remain bounded to the resin. This results in a cleaner NS3

helicase that requires one round of gel filtration chromatography rather than two, thus saving on time

and resources.

3.1.3 Comparison of yield between NS4B loop and NS3 helicase

It was seen that the yield of NS3 helicase was relatively lower than that of NS4B loop as NS3 helicase

band in Figure 2A was fainter than NS4B loop band in Figure 3A. This could be due to the relatively

higher induction temperature of 25°C carried out for NS3 helicase as compared to 18°C for NS4B loop.

Thus, the higher temperature could have lead to an overexpression of NS3 helicase, resulting in an

unbalanced ratio of protein to other cellular components in the bacteria [29-30]. This could then lead

partially folded NS3 helicase, exposing its hydrophobic sites. As a result, NS3 helicase became less

soluble and aggregated to from inclusion bodies (IB) [30-32]. Hence, less helicase was collected during

elution as the helicase within the IB were unable to bind to the nickel beads, causing it to be collected

together with other junk proteins during washing. Therefore, induction temperature should be at 18°C or

lower to minimise the loss of proteins due to inclusion bodies [33-34].

3.1.3 Comparison of yield between NS4B loop and NS3 helicase

From Figure 4A and 4B, total column, it can be seen that amount of NS3 helicase produced after

induction was the same as both bands have the same thickness. However, use of 4 ml of nickel beads to

purify the protein resulted in the collection of more proteins as compared to using 3 ml of nickel beads

as NS3 helicase band in Figure 4B elution comlumn is thicker than that in Figure 4A.

It was deduced that the amount of resin used affected the percentage yield of NS3 helicase. This could

be due to some junk proteins containing histidine tags/clusters, hence allowing them to bind to the

nickel beads. Other junk proteins could also bind to the beads by non-specific binding. However, due to

the stronger affinity between the thiodoxin and histidine tag on our protein and the nickel beads, our

protein would first bind to the beads. Subsequently, any excess beads remaining would attract junk

proteins, resulting in an impure elution.


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10-

50-

25-

20-

75-

250-

100-

150-‐

37-

(A)

M

Total

Load

F.T

E1

E2 E3 15-

kDa

D2 NS3 helicase

25-

15-

10-

20-

37-

M

Total

Load

F.T

E1 E2 E3

50-

75-

250-

100-

150-

(B) kDa

D2 NS3 helicase

Figure 4 SDS PAGE of (A) D2 NS3 helicase purification, loaded with 3 ml of beads and (B) D2 NS3 helicase, loaded with 4 ml of beads. M represents protein molecular marker; Total is the total cell lysate, load represents supernatant, F.T. is flow through (after incubation at 4°C) and E1, E2, E3 are the elution fractions. Thus, 4 ml of nickel beads were optimal for protein purification as it maximises the amount of protein

collected while minimising the junk proteins collected. This results were subsequently used for the

purification of NS4B loop.

4.Conclusions and Recommendations

Both D2 NS3 helicase and NS4B loop were successfully expressed and purified. Results showed that

NS4B displayed dissociation activities, with 4 ml of Ni2+ NTA resin being optimal for purification of

both NS3 helicase and NS4B loop. NS3 helicase purified by IMAC resulted in the collection of more

junk proteins as compared to NS4B loop, thus resulting in a lower percentage yield. From the yield of

both NS4B loop and NS3 helicase, it was deduced that induction should be carried out at 18°C or less to

minimise loss of proteins.

Purified D2 NS3 helicase and NS4B loop could be used for further binding studies to understand about

the binding interfaces. Binding studies include nuclear magnetic resonance titration discover the sites

where specific interactions between the two proteins take place, as well as isothermal titration

calorimetry, which measures extent of binding between small molecules and the interaction sites found

by NMR titration. Crystalline protein structures could also be explored by X-ray crystallography to

visualise plausible interactions between the two proteins D2 NS3 helicase and NS4B loop or other

ligands. Determination of the binding interface between NS4B loop and NS3 helicase would provide

insights for development of chemicals that interfere with these binding interactions for development of

targeted drugs against DENV.


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Yishun Junior College_Lou Wei Hao Darren_ SSEF report_BC020

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