Alex Coronado

27 Oct 2015
MCB 4021C

Materials and Methods

Recombinant Protein Expression
To produce recombinant DNA for protein expression, BL21 competent E. coli were used.
The pET102-BasTM gene (1 L) was transformed into 50 L of BL21 cells by
incubating on ice for 15 minutes and then at 42*C for 30 seconds. Then, 250 L of SOC
medium was added to the cells and they were incubated overnight at 37*C while
shaking at 200 rpm. The transformation reaction was added to LB medium containing
cabenicillin and then incubated for 3 hours at 37*C. Then, 50 L of the Lac repressor
Isopropyl-b-D-thiogalactopyranoside (IPTG) was added and then incubated overnight.
The recombinant protein was then purified. First, a bacterial cell pellet that weighed 0.31
grams was generated by centrifuging at 3000 rpm for 15 min. The pellet was solubilized
with 6.2 mL of X-tractor buffer, 62 L of lysozyme and 12.4 L of DNase I. The mixture
was placed on a rocker and incubated at room temperature for 20 minutes. The
bacterial lysate was then centrifuged for 20 minutes at 10,000 rpm and the supernatant
containing the cleared bacterial protein lysate was collected in a tube.
To separate the proteins, affinity chromatography was performed. The cleared bacterial
protein lysate was combined with prewashed resin that was prepared using 1 mL of
TALON His-tag resin and 5 mL of wash buffer and was incubated while shaking at room
temperature for 20 minutes. Then the mixture was centrifuged for 5 minutes at 700 x g
and the pellet was washed 3 times and resuspended before transferring to a purification
column. Elution buffer containing imidazole was added to the column and ten 0.5 mL
fractions were collected. The fractions were then analyzed my measuring absorbance at
280 nm, and a standard curve was used to determine concentration.
ELISA
An ELISA (enzyme-linked immunosorbent assay) was performed. The anti-MCT1
capture antibody was coated to a well plate and incubated overnight at 4*C allowing the
antibodies to bind to the plastic well. The well plate was set up with 3 columns. Column
1 was for PBS only, column 2 was for the control 6XHis, and column 3 was for the BasTM 6XHis. Any unbound antibody was removed and all wells were washed 3 times with

PBS-T (detergent). Working with 100 L aliquots, and column 1 always receiving PBS,
bovine serum albumin (BSA) was added to columns 2 and 3. The plate was then
incubated for 30 minutes at 37*C. Wells were washed 3 times, and every time after
incubation. Then the mouse brain proteins (diluted 100 L/mL in PBS) were added to
columns 2 and 3 and incubated for 30 minutes at 37*C. Next, control 6XHis was added
to column 2 and recombinant protein Bas-TM 6XHis was added to column 3, then
incubated and under the same conditions as pervious steps. The primary antibody anti6XHis (diluted 1:500 in PBS) was added to columns 2 and 3 and then incubated (30
minutes at 37*C). After washing, the secondary antibody (diluted 1:1000 in PBS) was
added, which was alkaline phosphate-conjugated goat anti-mouse, and then incubated.
Last the AP substrate was added to all the rows which converted the substrate yellow.
The reaction was stopped by adding 50 L of 2N NaOH and the absorbance was read
at 405 nm.
Results
The standard curve used to find the unknown concentrations is below in Figure 1. A
logarithmic tread line was used. The concentration for the first unknown was 0.210
mg/mL and for the second unknown was 0.218 mg/mL. For the brain, the protein
concentration was 0.567 mg/mL.
1.2
1.1
1
0.9

f(x) = 0.42 ln(x) + 0.76

R = 0.97

0.8
0.7

Absorbance at 595 nm

0.6
0.5
0.4
0.3
0.2
0.1
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

Protein Concentration (mg/mL)

Figure 1 Standard curve for BSA concentration.

The average absorbance for each experimental condition of the ELISA binding assay is
graphed below in Figure 2.
0.18
0.16
0.14
0.12
0.1

Absorbance at 405 nm

0.08
0.06
0.04
0.02

BasTM
6XHis

Control
1

6XHis

Figure 2 Bar graph representing the absorbance values of BasTM 6XHis and Control
6XHis. The standard deviation for BasTM 6XHis was 0.0292 and for the control was
0.1045.
A paired one-tailed T-test resulted in a p-value of 0.235.