Nanjing Xinfan Bio: Experimental steps and precautions for GST fusion protein expression and purification - Database & Sql Blog Articles

GST expression

Fusion protein carrier

pGEX-KG

Size: 5006bp, ampicillin resistance (Ampr),

Iptg induction

expression

Enzyme

Cut sites: BamHI 930, SmaI 937, EcoRI 962, XbaI 966, NcoI 974, SalI 980, XhoI 985, SacI 992, HindIII 994

GST

Molecular weight

:

Construct pGEX-KG-YFG reorganization

Plasmid

1. Analyze the genes of interest (your favorite)

GENE

, YFG)

Primer Premier 5.0 software, which analysis of YFG containing restriction sites, pay attention to whether it overlaps with the multiple cloning site of pGEX-KG vector

2, determine the appropriate double enzyme cleavage site

NEB website () Double Digest Finder software to find the best double cut combination (the following table)

NEB double restriction enzyme map BamHI EcoRI NEBuffer EcoRI + BSA at 37 ° C. BamHI may exhibit star activity in this buffer. XbaI NEBuffer 3 + BSA at 37 ° C. At least one enzyme has < 100% activity in this buffer, so additional NcoI NEBuffer 3 + BSA at 37°C. SalI NEBuffer 3 + BSA at 37°C XhoI NEBuffer 3 + BSA at 37°C. SmaI XbaI NEBuffer 4 + BSA at 25 °C with SmaI, then add XbaI and raise temperature to 37°C. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. NcoI NEBuffer 4 at 25° C with SmaI, then add NcoI and raise temperature to 37°C. XhoI NEBuffer 4 + BSA at 25°C with SmaI, then add XhoI and raise temperature to 37°C. SacI NEBuffer 4 + BSA at 25°C with SmaI, Then add SacI and raise temperature to 37°C. HindIII NEBuffer 4 at 25°C with SmaI, then add HindIII and raise temperature to 37°C. At least one enzyme has < 100% activity in this Buffer, so additional units of enzyme and/or longer incubation time may be necessary. EcoRI NcoI NEBuffer EcoRI at 37°C. SalI NEBuffer EcoRI + BSA at 37°C. XhoI NEBuffer EcoRI + BSA at 37°C. SacI NEBuffer 1 + BSA at 37°C. EcoRI may exhibit star activity in this buffer. HindIII NEBuffer EcoRI at 37°C. XbaI NcoI NEBuffer 2 + BSA at 37°C. SalI NEBuffer 3 + BSA at 37°C. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. XhoI NEBuffer 2 + BSA at 37°C. SacI NEBuffer 4 + BSA at 37°C. This buffer is not supplied with either enzyme .
At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. HindIII NEBuffer 2 + BSA at 37°C. NcoI SalI NEBuffer 3 + BSA at 37°C. XhoI NEBuffer 2 + BSA at 37°C. SacI NEBuffer 1 + BSA at 37°C. HindIII NEBuffer 2 at 37°C. SalI XhoI NEBuffer 3 + BSA at 37°C. XhoI SacI NEBuffer 1 + BSA at 37°C. At At least one enzyme has <100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. HindIII NEBuffer 2 + BSA at 37°C. SacI HindIII NEBuffer 2 + BSA at 37°C. One enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.

Control YFG, vector multiple cloning site, determine the upstream and downstream enzyme cleavage sites

3. Design PCR upstream and downstream primers

Primer Premier 5.0 software for designing PCR upstream and downstream primers

Up to 6 bases outside the restriction site

The 3' end is not A, preferably G or C, but it is not recommended to use GC or CG to end

At least 3 bases are fully paired at the 3' end

Rating is greater than 70

[note]

Upstream primer: Whether to add appropriate bases to ensure that the open reading frame is not disturbed

Downstream primer: Add stop codon (UAA, UAG, UGA)

4. Primer synthesis and preservation

Synthesis: Shanghai Shenggong Bioengineering Technology Service Co., Ltd. (Email:, Tel); Purification method: column chromatography or polyacrylamide gel electrophoresis; price 1.30/base

Storage: Storage concentration: 100pmol/μl (100μM), working concentration: 10pmol/μl (10μM), stored at -20°C

5. PCR amplification of YFG

Template: plasmid 10 ng / μl diluted a small amount -20 ° C preservation

Primer: 10pmol/μl (10μM) -20°C preservation

Taq enzyme: NEB Quick-Load Taq 2×Master Mix Amplified fragment less than 2.0kb

Reaction system (on ice when prepared)

25 μl reaction system 50 μl reaction system template 1 μl 2 μl upstream primer 1 μl 2 μl downstream primer 1 μl 2 μl 2× Master Mix 12.5 μl 25 μl Deionized H ₂ O 9.5 μl 19 μl

Reaction conditions

(1) Pre-denaturation 94 ° C 5 min

(2) Denaturation 94 ° C 30 s

(3) Annealing to be determined for 30 s

(4) Extended 72 ° C to be determined

(5) Repeat 2-5 25-30 cycles

(6) Filling the gap 72 ° C 10 min

(7) Temporary storage 10 ° C

[note]

Annealing temperature: Refer to 4(G+C)+2(A+T)-4 (complementary base), refer to Ta Opt (Primer Premier 5.0)

Extension time: Taq enzyme: 1kb/min

Reduce the number of cycles by less than 30

Detection of PCR products by agarose electrophoresis

0.8% effective separation range: 10~0.8kb; 1.0% effective separation concentration 7~0.5kb

Add 5μl EB mother liquor (5mg/ml) to 50ml TAE

100V, 30-45min

Photographing or recycling of glue under UV light

6, build pGEX-KG-YFG

Digestion: double digestion PCR product, pGEX-KG

Recycling: direct recovery of PCR products, gel removal after pGEX-KG electrophoresis

Connection: pGEX-KG 50ng, insert 150ng

Conversion plating: Ampr

Pick a single: Ampr (four colonies are enough)

Identification: small plasmid digestion or bacterial PCR

7. Transformation of BL21(DE3)pLysS strain to detect the expression of GST fusion protein

(1) Melting BL21(DE3)pLysS competent cells on ice (Tiangen)

(2) In a 2 ml centrifuge tube, add 25 μl of BL21 + 3 μl plasmid (300-500 ng) and mix (plasmid ≤ competent 1/10)

(3) Place on ice for 30min

(4) 42 ° C, 90 s

(5) Place on ice for 2-3min

(6) Add 300μl LB (no antibiotics) (equivalent to 10 times the volume of LB), 37 ° C, 250 rpm, 1 h

(7) 4000 rpm, 2 min, discard the supernatant, and mix the other plates after plating (Ampr), 37 ° C, overnight (16 h)

(8) Pick monoclonal colonies, 5 ml LB (Ampr), 37 ° C, 250 rpm, overnight (16 h)

() Dilute 10 times, 20 times, 50 times to measure OD600

() Select the appropriate dilution factor, 5ml bacterial solution, 37 ° C, 250 rpm, 1 h, determine OD600

(7) Take 100 μl of bacterial solution and add 5 ml of LB (Ampr) (50-fold dilution), 37 ° C, 250 rpm, overnight (16 h)

(8) Take 500 μl of bacterial solution and add 5 ml of LB (Ampr) (10-fold dilution) (the remaining bacterial solution is stored at 4 ° C), and measure OD600.

(9) 37 ° C, 250 rpm, 2 h (OD600: 0.6-0.8), OD600

(10) Place at room temperature for 20 min (shaker cooling, 30 ° C)

(11) Take 100 μl as a pre-induction control and place on ice

(12) Add IPTG to the bacterial solution to a final concentration of 0.5-1 mM

(13) 30 ° C, 250 rpm, 2 - 4h (can do time gradient), measure OD600

(14) Take 100 μl as the post-induction control, together with the pre-induction bacterial solution, centrifuge at 12000 rpm for 2 min, and add 50 μl of 1×SDS loading buffer.

(15) Take 4ml of bacterial solution, centrifuge at 12000rpm for 2min, and collect it in 2ml centrifuge tube.

(16) Resuspend the cells by adding 1 ml of GST lysis buffer

(17) Ultrasonic broken cells: Ultrasound 20s, interval 10s, 3-5 times, ultrasonic intensity 200-300W (ice bath)

(18) After the ultrasonic solution, the bacterial liquid was exchanged into a new 1.5 ml centrifuge tube, centrifuged at 4 ° C, 12000 rpm for 5 min.

(19) Supernatant supernatant: Take 25 μl of supernatant and add 25 μl of 2×SDS loading buffer (the remaining supernatant is stored at -80 ° C)

(20) Post-ultrasound precipitation: The pellet was resuspended in 1 ml of GST lysis buffer, 25 μl was added, and 25 μl of 2×SDS loading buffer was added (the remaining pellet was stored at -80 ° C).

(21) Four samples were boiled for 3 min, 12000 rpm, and centrifuged for 5 min.

(22) 8-10% SDS-PAGE, 30 μl loading, sequence: pre-induction of the cells, induction of the cells, post-ultrasound supernatant, post-ultrasonic precipitation

(23) Coomassie blue staining

[note]

(1) The OD value of the bacterial solution can be less than 1.

(2) It is best to make a gradient for induction time, 2-6h

(3) The induction temperature is properly explored, 24 ° C, 30 ° C

(4) IPTG concentration can be gradient, 1 mM

(5) The ultrasonic condition can be changed according to the actual situation, as long as the bacteria are fully lysed, that is, the bacterial liquid is clear and not sticky.

8, sequencing confirmation

Shanghai Shenggong Bioengineering Technology Service Co., Ltd. (Tel)

Sequencing primers: pGEX-3 universal primer

Sequencing samples: 1 ml of overnight bacteria; plasmid (concentration greater than 50 ng/μl, 10 μl)

9, the middle of the plasmid (Promega)

10. Expression of purified GST fusion protein

(1) The transformed bacterial liquid, or reconverted BL21(DE3)pLysS

(2) Activation: 20 μl of bacterial solution was added to 11 ml of LB (Ampr) (500-fold dilution), 37 ° C, 250 rpm, overnight (16 h)

(3) Add 10 ml of bacterial solution to 100 ml of LB (Ampr) (10-fold dilution) (the remaining bacterial solution is stored at 4 ° C)

(4) 37 ° C, 250 rpm, 1 h 10 min-1 h 20 min, OD600 0.6-0.8 (OD600 less than 1.0)

(5) Take 1ml of bacterial liquid as a pre-induction control, place on ice

(6) Add IPTG to a final concentration of 1 mM

(7) 30 ° C, 250 rpm, induction of appropriate time (pre-experiment determination)

(8) Take 1ml of bacterial liquid as the post-induction control, together with the pre-induction bacterial solution, centrifuge at 12000 rpm for 2 min, collect the cells, and add 100 μl of 1×SDS loading buffer.

(9) The remaining bacterial liquid is divided into two parts, poured into a 50 ml centrifuge tube, centrifuged at 5000 rpm for 20 min, and the cells are collected.

(10) Resuspend the cells by adding 8-10ml GST lysis buffer per tube and transfer to a small beaker (no bubbles)

(11) Ultrasonic broken cells: ultrasound 3s, interval 10s, 40 times, ultrasonic intensity 200-300W (ice bath)

(12) Transfer the ultrasonicated bacteria solution to a 50ml centrifuge tube, centrifuge at 20 °C for 10-30 minutes at 4 °C, 13000 rpm.

(13) Transfer the supernatant to a 50 ml centrifuge tube, take 50 μl, and add 50 μl of 2×SDS loading buffer (the remaining supernatant is stored at -80 ° C)

(14) The pellet was resuspended in 16-20 ml of GST lysis buffer (or PBS), 50 μl was taken out, and 50 μl of 2×SDS loading buffer was added (the remaining pellet was stored at -80 ° C).

(15) Boil four samples for 3 min, 12000 rpm, and centrifuge for 5 min.

(16) 8-10% SDS-PAGE detection of induction, ultrasound is appropriate, 30μl loading, sequence: induced pre-bacteria, induced bacteria, post-ultrasound supernatant, post-ultrasound precipitation

(17) Coomassie blue staining

(18) Mix glutathione sepharose beads (4B) (mixed slurry), add 200 μl to a 15 ml centrifuge tube (2 parts)

(19) Add 10ml PBS, mix, 3000rpm, centrifuge for 3min, discard the supernatant

(20) Add the supernatant after sonication, gently shake at 60 °C for 4 minutes (or overnight) (horizontal)

(21) 4 ° C, 3000 rpm, centrifugation for 3 min

(22) Wash 2 times with 10ml GST Lysis Buffer (on ice)

(23) Wash 2 times with 10ml TBS (containing 5mM MgCl2, 1mM DTT) (on ice)

(24) Discard the supernatant and add 1 column bed volume of TBS (containing 5 mM MgCl2, 1 mM DTT, 50% glycerol), and mix.

(25) Take the suspension to determine the protein concentration or SDS-PAGE

(26) -20 ° C save beads

11, GST-Pull-down

(1) Untreated cells or transfected cells in a 10 cm culture dish (3-5 μg plasmid transfected with 10 cm culture dish, Cos-7, 293T or other cells, transfected for 24 h)

(2) Add 1 ml of cell lysis buffer, and scrape the cells with cells (on ice)

(3) Collect the lysate into a 1.5 ml centrifuge tube, shake for 30 s, place on ice for 5 min, repeat 2-3 times, fully lyse the cells.

(4) 4 ° C, 12000 rpm, centrifuge for 15 min, collect the supernatant

(5) Determine the protein concentration and take 1mg of total protein for GST-Pull-down?

(6) Leave 30 μl as the input control (the amount of input and Pull-down protein is about 1/10)

(7) The remaining solution, add 20 μl of glutathione sepharose beads (washed with PBS), shake at 4 ° C for 20 min.

(8) 12000 rpm, centrifuge for 3 min

(9) Collect the supernatant, divide it into two parts, add 1-15μg GST-bound beads and add the same amount of GST-fusion protein bound beads, 4°C, gently shake for 60min.

(10) 3000 rpm, centrifuge for 3 min, recover the supernatant

(11) 1 ml of cell lysis buffer washed 3 times

(12) Discard the supernatant and add 25μl 2×SDS loading buffer

(13) boil for 3 min, 12000 rpm, centrifuge for 3 min

(14) SDS-PAGE, loading sequence: cell lysate input, x, GST, x, GST-fusion protein (x: LSB)

(15) Coomassie blue staining or immunoblotting

Elisa kit, imported elisa kit, elisakit, imported serum, imported reagent, gelatin zymography test kit, rat elisa test kit, human elisa test kit-Nanjing Xinfan Biotechnology Co., Ltd.