Ohanin a novel protein from king cobra (ophiophagus hannah) venom 2

181

APPENDICES


























Figure A.1 Map of pGEM

®
-T Easy Vector



182













Figure A.2 Production of recombinant proteins. (a) The expression vector contains the
lac promoter which drives the expression of lacZ gene encoding for β-
galactosidase. Lactose or its analog IPTG will stimulate the expression of β-
galactosidase. (b) If lacZ is replaced by the gene encoding the protein of interest, lactose
or IPTG will stimulate the expression of desired proteins.
Many proteins of interest are expressed in low concentration in endogenous system.
Through recombinant DNA technology as above, large quantities of recombinant proteins
can be produced using an in vitro expression system.
(Modified from
Note
Blue white selection: Many vectors (such as pUC series) carry coding information for

the first 146 amino acids of the β-galactosidase gene. Embedded in this coding region is
the multiple cloning site (does not disrupt the reading frame of the gene) into which the
insert DNA is cloned. After IPTG induction, this 146 amino acid fragment of β-
galactosidase protein is expressed but incapable of acting on the chromogenic substrate
(X-gal). It is only when transformed in an appropriate host cells which expressed the
other half of the carboxyl terminal fragment of the β-galactosidase protein, these two
protein fragments can then associate to form an enzymatically active protein to cleave the
chromogenic substrates. This process is called as α-complementation. Properly expressed
β-galactosidase protein (after α-complementation) will turn cells into blue colonies when
plated on plates containing X-gal.

If the insert DNA is cloned in the multiple cloning sites and disrupted the reading frame,
amino terminal fragment of β-galactosidase protein is not produced. Hence, colonies will
remain white. However, if the inserts do not disrupt the reading frame of LacZ gene, there
is still a possibility to have blue colonies harboring the desired inserts.

183
Table A.1 Reagents and chemicals used for molecular biology experiments.


Name Components Amount in 1 l Note

TAE buffer (10X)

Tris-base
EDTA

0.40 M
0.01 M
Æ Prepare from the stock

solution of the chemicals, adjust
to pH 7.8 using glacial acetic
acid and autoclave
Æ Dilute to 1X before use

SOB medium
(for preparation of
competent cells)

Tryptone
Yeast extract
NaCl
KCl
MgCl
2
.6H
2
O
MgSO
4
.7H
2
O

20.00 g
5.00 g
0.58 g
0.19 g
2.03 g
2.46 g

Æ Prepare without Mg
2+
, adjust
to pH 7.0 and autoclave
Æ A 2 M stock of Mg
2+
(1 M
MgCl
2
and MgSO
4
, filter
stelize) is used to make the
medium 20 mM in Mg
2+

TB buffer
(for preparation of
competent cells)

Pipes
MnCl
2
CaCl
2
KCl

10 mM
55 mM
15 mM

25 mM
Æ Prepare from the stock
solution of the chemicals, adjust
to pH 6.7 and filter sterilize

IPTG stock
(20 mg/ml)
IPTG 2.00 g

Æ Dissolve in dH
2
O, filter
sterilize and store at -20
o
C
Æ IPTG induces synthesis of a
β-galactosidase. It is used to
detect lacZ gene expression in
cloning experiment
Æ Use for blue-white colonies
selection

X-gal stock
(50 mg/ml)

X-gal 5.00 g Æ Dissolve in dimethyl
formamide, filter sterilize, wrap
containers with aluminium foil
and store at -20
o

C
Æ Use as chromogenic
substrates in blue-white
colonies selection





184
Table A.2 Antibiotics, reagents and chemicals used for growing bacterial culture and
for recombinant protein expression.


Name Components Amount in 1 l Note

LB Medium Tryptone
Yeast extract
NaCl
10.00 g
5.00 g
10.00 g
Æ Adjust to pH 7.5, sterilize by
autoclaving
Æ To make the agar medium, add
15 g agar to 1 l liquid medium
before autoclaving

Ampicillin (Amp)
(100 mg/ml)

Ampicillin 100.00 g Æ Dissolve in dH
2
O, filter
sterilize and store at -20
o
C
Æ Use for inhibition of cell-wall
synthesis by interfering with
peptidoglycan cross-linking

IPTG stock
(1 M)

IPTG 238.30 g Æ Dissolve in dH
2
O, filter
sterilize and store at -20
o
C
Æ Use to induce expression of
plasmid-based genes for the
production of recombinant
proteins under control of the lac
promoter

Lysozyme
(100 mg/ml)

Lysozyme 100.00 g Æ Dissolve in dH
2

O, filter
sterilize and store at -20
o
C
Æ Use for hydrolyzing
peptidoglycan. Osmotic lysis will
occur as a result of destruction of
the bacterial cell wall.



185
Table A.3 Solutions for preparing 15 % resolving and 4 % stacking gels for SDS-
glycine Polyacrylamide Gel Electrophoresis (SDS-PAGE).


Solutions components Component volumes/
4 gels
(0.75 mm thickness)
Note

Resolving
H
2
O
30 % Acrylamide mix (37.5:1)
1.5 M Tris-base (pH 8.8)
10 % SDS
10 % Ammonium persulfate
TEMED

4.70 ml
10.00 ml
5.00 ml
200.00 µl
100.00 µl
10.00 µl

Æ Mix the components in
the order shown, swirl the
mixture rapidly and pour the
resolving mixture into the
gap between glass plate

Stacking
H
2
O
30 % Acrylamide mix (37.5:1)
0.5 M Tris-base (pH 6.8)
10 % SDS
10 % Ammonium persulfate
TEMED


6.00 ml
1.32 ml
2.52 ml
100.00 µl

50.00 µl
10.00 µl

Æ Mix the components in
the order shown, swirl the
mixture rapidly and
pour the stacking mixture
onto the surface of the
polymerized resolving gel
Æ Insert a clean Teflon
comb into the stacking
solution




186
Table A.4 Reagents and chemicals used for SDS-PAGE.


Name Components Amount in 1 l Note

SDS-glycine
buffer (5X)
Tris-base
Glycine
SDS
15.10 g
72.00 g
5.00 g

Æ Dissolve Tris and Glycine,
adjust pH to 8.3
Æ Add SDS and adjust the
volume to 1000 ml
Æ Dilute to 1X before use

Staining solution

Coomassie
Brilliant Blue
R-250
Ethanol
Acetic acid
1.00 g


300.00 ml
100.00 ml

Æ Dissolve Coomassie Brilliant
Blue R-250 in 600 ml dH
2
O,
ethanol and acetic acid

Destaining
solution

Methanol
Acetic acid

400.00 ml
100.00 ml
Æ Mix 500 ml dH
2
O with
methanol and acetic acid

Gel drying solution

Methanol
Acetic acid
Glycerol
400.00 ml
100.00 ml
100.00 ml

Æ Mix 400 ml dH
2
O with
methanol, acetic acid and glycerol






Table A.5 Preparation of SDS gel-loading buffer.


Name Components Amount Note

SDS gel-
loading
buffer (4X)


Bromophenol blue
Glycerol
Upper Tris (pH 6.8)
20 % SDS

0.0185 g
8 ml
10 ml
4 ml

Æ 4X SDS gel-loading buffer
without the β-mercaptoethanol can
be store at room temperature
Æ Add 28 µl of β-mercaptoethanol
to 190 µl of loading buffer before
use, store the remaining loading
buffer with β-mercaptoethanol at -
20
o
C after use

187
Table A.6 Preparation of DNA ladders for molecular biology experiments.


Components Note

DNA ladders stock
(1 kb plus, 100 bp, 50 bp)
(Working stock: 1 µg/ µl)

Æ Mix 10 µl of stock DNA ladders with 90 µl
dH
2
O and 20 µl loading dye, store at -20
o
C
Æ Load 6 µl each time