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Recombinant DNA Techniques (DNA Cloning) PowerPoint PPT Presentation


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生命科學實驗. Plasmid DNA miniprep . PCR R. E. digestion Gel purification, Ligation, Transformation Preparation of competent cells. Gene Plasmid b.p . ORF E.coli strain. Recombinant DNA Techniques (DNA Cloning). GST-LEU2 SDS-PAGE Coomasie Blue ( Green Angel) Staining

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Recombinant DNA Techniques (DNA Cloning)

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Recombinant dna techniques dna cloning

生命科學實驗

Plasmid DNA miniprep.

PCR

R. E. digestion

Gel purification, Ligation, Transformation

Preparation of competent cells

Gene

Plasmid

b.p.

ORF

E.coli strain

Recombinant DNA Techniques (DNA Cloning)

GST-LEU2

SDS-PAGE

Coomasie Blue (Green Angel) Staining

O.D.280, Bradford and Bicinchoninic acid assays (BCA)

Expression, Purification and quantification of recombinant protein

M.W.

Ab

Detection of recombinant protein

Immunological (Western) Analysis

Application of recombinant protein

Enzyme assay (DHase) of GST-LEU2


Recombinant dna techniques dna cloning

Outline for expression and purification of GST-fusion protein

Clone gene into pGEX vector

Week 1

-/ + IPTG induction

SDS-PAGE

Test expression of fusion protein (small scale),

SDS-PAGE

Week 2

Lysozyme,

DNaseI/ sonication

Culture (large scale), harvest and lyse cells

Binding

Washing

Elution

Week 3

GST-fusion protein purification,

making new protein gel for week 4

Week 4

SDS-PAGE

Coomasie Blue Staining

Bradford assays

Detection and quantification of GST fusion protein


Recombinant dna techniques dna cloning

GST-fusion system

  • A one-step system to purify protein.

  • Developed by Smith and Johnson in 1988, using glutathione S-transferase (GST) protein from Schistosomajaponicum.

  • Protein of interest is usually fused at the C-terminus of GST.

  • GST binds to glutathione, an tripeptide antioxidant, with high affinity.

beads

GST protein

glutathione


Recombinant dna techniques dna cloning

IPTG Induction

  • A way to induce expression of large amount of protein in E.coli.

tac promoter: hybrid promoter of trp and lac, strong expression upon induced

X

Lac repressor

GST

LEU2

P

L

:IPTG

conformational change of Lac repressor

P

L

GST

LEU2


Recombinant dna techniques dna cloning

SDS-PAGE

(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

聚丙烯胺膠體


Recombinant dna techniques dna cloning

Expression of GST-LEU2 Protein

GST

GST-LEU2

GST-leu2

IPTG (h):

0

0

0

3

3

3

5

5

5

Green Angel Protein Staining

170

130

95

72

55

43

34

26

17


Recombinant dna techniques dna cloning

GST-LEU2 protein purification (large scale)

Lysate preparation (1.5 hours)

Glutathione-beads binding (2 hours)

Wash (40 mins)

Elution (40 mins)


Gst leu2 lysate preparation

GST-LEU2 lysatepreparation

Lysis buffer:

50 mMNaCl: providing a osmotic shock and keep protein soluble

50 mMTris HCI, pH 7.5

1 mM EDTA: inhibits divalent cation-dependent proteases

1% (v/v) Triton X-100: detergent, increasing protein solubility

Protease inhibitor cocktail

PMSF (phenylmethylsulfonyl fluoride): serine protease inhibitor,

rapidly degraded in water

Lysozyme: a glycoside hydrolase of peptidoglycans, break the carbohydrate in the bacterial cell wall.

DNase I: digest bacterial DNA to reduce viscosity of lysate

Other lysis methods:

Sonication: useful for a variety of cell types.

Homogenization/ Glass bead/ French press: high pressure to force cells through a narrow space, thereby shearing the cell walls and membrane.


Gst leu2 purification

GST-LEU2 purification

beads

+

GST-LEU2

lysate

beads

GST-LEU2

after-binding

washing

beads

GST-LEU2

wash


Gst leu2 elution

GST-LEU2 elution

beads

GST-LEU2

free glutathione

beads

GST-LEU2

elute


Gst fusion protein purification

GST fusion protein purification

Lane 1: whole cell lysate

Lane 2: after binding

Lane 3: wash

Lane 4: elute


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