The sperm whale Mb mutants were constructed by cassette mutagenesis. The
sequence is from NCBI. The expression vector of wild-type sperm whale Mb was from Hung-Ming Lin.16 The cassette including silent EcoR V restriction site was inserted between the BamH I and Nco I sites. The expression was in Escherichia coli strain pET-28a(+).
The mutations of MbL29X/H64D/V68L/I107M, MbH93X, MbF43Y, MbF46Y, and MbV17W were constructed by Site-Directed mutagenesis strategies.
(1) Primer design:
Table 3. Primer design for site-saturated mutagenesis
The letters with gray background in the sequence line of primers show the target mutations, and “N” means A, T, C, G four bases, which are arranged in order to form 20 possible amino acids. The bolded letters indicate silent mutation for EcoR V mapping analysis and these constructions are marked with underline.
(2) QuikChange PCR:
Reagent Volume(µL)
Template 1 Primer 1 (10 μM/μl) 1
Primer 2 (10μM/μl) 1
10X Pfu Buffer 5
dNTP (10 mM) 4
DDW 37
Pfu polymerase 1
Total reaction volume 50
Table 4. QuikChange Site-Directed Mutagenesis Kit PCR composition
segment Cycles Temperature time
1 1 95 ℃ 2 min
2 18
95 ℃ 30 sec
Primer Tm – 5~8 ℃ 30 sec
72 ℃ 12 min
3 1 72 ℃ 10 min
4 1 4 ℃ ∞
Table 5. QuikChange Site-Directed Mutagenesis Kit program
(3) Dpn I digest parental DNA template:
The digest reaction was incubated at 37 ℃ for 3 hours to digest the parental supercoiled DNA.
Reagent Volume(µL)
PCR products 17
10X NEBuffer 4 1
DDW 1
Dpn I 1
Total reaction volume 20
Table 6. QuikChange Site-Directed Mutagenesis Kit PCR products digestion
(4) Transformation into XL1-Blue and enzyme mapping:
The digestion of QuikChange products were added into 100 µl E.coli XL1-Blue competent cells of each reaction and incubated on ice for 20 min. The cells were transformed by heatshock methods for 1 min at 42 ℃, following 1 min on ice. Then, the cells were transferred to 1 ml LB medium immediately and shaken at 200 rpm for 1 hour at 37 ℃ incubator. After that, the cells were centrifuged at 8,000 rpm for 1 min and propagated on LB plate containing 25 µg/ml kanamycin. These plates were incubated at 37 ℃overnight. Selected and cultured the transformed colonies in 3 ml LB medium containing 25 µg/ml kanamycin at 37 ℃overnight. The plasmid DNAs were isolated by Plasmid Miniprep Purification Kit, according to the manufacturer instructions.
(5) Sequencing analysis of myoglobin mutants:
The exact amino acid substitution at Val17, Leu29, Phe43, Phe46, and His93 positions were determined by sequencing of the DNA using ABI PRISM 3100 auto-sequencer. Nucleotide sequencing was performed using the dideoxynucleotide chain-termination method with only one forward of reverse primers. Sequencing reactions were carried out with BigDye® Terminator v3.1 Cycle Sequencing Kit, according to manufacturer protocol. Each of the samples were performed with 1 µl each forward or reverse primer, 2 µl plasmid DNA, 3 µl 5X sequencing buffer, 1 µl premix and ddH2O to get a final volume of 20 µl. Each of reactions was performed on ABI PRISM® 3100 Genetic Analyzer, following the manufacture’s guidelines.
2-2-3 Preparation of Clones
The plasmids after checked were added into 100 µl E.coli BL21 (DE3) competent cells of each reaction mixture and incubated on ice for 20 min. The cells were transformed by heatshock methods for 1 min at 42 ℃, following 1 min on ice.
Then, the cells were propagated on LB plate containing 25 µg/ml kanamycin with 40 µl. These plates were incubated at 37 ℃ overnight. Injected the colonies and cultured in 1ml LB medium containing 25 µg/ml kanamycin at 37 ℃ overnight.
Taking one of the medium mixed the same volume of glycerol and stocked at -80 ℃.
2-2-4 Preparation of SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
The glass-plate sandwich of the electrophoresis apparatus was assembled according to manufacturer’s instructions. The gel solution was prepared as follows:
Solution Separating gel (20%)
ddH2O 2 ml
30% acrylamide mix 6.6 ml 1.5 M Tris ( pH 8.8) 2.5 ml
20% SDS 50 µl
10% ammonium persulfate 200 µl
TEMED 10 µl
Solution Stacking gel (4%)
ddH2O 3 ml
30% acrylamide mix 0.67 ml 1 M Tris ( pH 6.8) 1.25 ml
20% SDS 50 µl
10% ammonium persulfate 50 µl
TEMED 10 µl
Table 7. 20% separating gel and 4% stacking gel of SDS-PAGE
2-2-5 Protein detection
For expression experiments, protein detection was competed with SDS-PAGE and Western blotting methods. All the clones were inoculated in 3 ml LB medium containing 25 µg/ml kanamycin at 37 ℃ overnight. The cells were centrifuged at 8,000 rpm for 1 min and re-suspended with 200 µl lysis buffer. After that, the solutions were immediately frozen in liquid nitrogen, then defrosted at 42 ℃ water
bath and repeated for several times. The solutions were centrifuged at 13,000 rpm for 1 min. The 20 µl supernatant was mixed with 5X sample treatment buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol , and 0.05%
bromophenol blue), and heated at 100 ℃ for 10 min. Electrophoresis was performed according to the manufacturer’s instructions. After electrophoresis, the gel was soaked in Coomassie Blue R 250 staining solution for 20 min, and the gel was destained with the destaining solution I (40% methanol, 7% acetic acid) and II (5% methanol, 7%
acetic acid) until the stained band was distinct against a clear background.
In Western blotting experiments, the supernatants were electrophoretically separated with SDS-PAGE and transferred onto polyvinylidene difluoriede (PVDF) membranes. The PVDF membranes were washed by the PBS buffer (pH 7.6) containing 0.05% Tween 20 (PBST) for 10 min, and immunodetection was carried out by following the procedure for the ECL Western blotting system, using monoclonal antibody raised against the hemolysin (1:500) and anti-mouse immunoglobulin HRP-peroxidase conjugate (1:5000). Excess ligand was washed away with PBST for 30 min, and the detection of the proteins was performed according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Membranes were exposed to Hyperfilm ECL (Amersham) for different times or until a suitable signal was obtained from the exposed films.
Inoculate