Gene Expression and Regulation

•Transient or stable expression

•Controlled regulation of expression

•Tet-off and Tet-on system

•pQE system: selection of vector, host, fusion tag,regulation, purification strategy

Lecture 16

Transient transfection: Depending on cell types, upto 80% of a population of cells can express the transfected gene in a transient fashion. This allows functional assay, gene reporter assay, and other studies.

Stable transfection: After entering the cell, DNA undergoes a series of nonhomologous recombination to form a large concatemeric structure that eventually integrates into the chromosome. In the best cases, 1 in 1000 in the original transfected population stably expresses the gene from the vector.

Transient vs. Stable Expression

Intrinsic GFP

Anti-GFP

Bar, 25 µm

AgeI/PinAI 601NheI 592

NdeI 235

BsrGI/SspBI 1323BglII (X in GW/G6) 1340XhoI (X in G14) 1345SacI 1348HindIII 1353

KpnI 1676

XbaI 1858HindIII 1863

EcoRI 1870PstI 1875KpnI 1886BamHI 1901XbaI 1912

NdeI 235NheI 592AgeI/PinAI 601

BsrGI/SspBI 1323BglII (X in GW/G6) 1340XhoI (X in G14) 1345SacI 1348HindIII 1353

KpnI 1676

XbaI 1858HindIII 1863

EcoRI 1870PstI 1875KpnI 1886BamHI 1901XbaI 1912

EcoO109I 4360

AflIII 5180

CMV IE

EGFP

eIF-5A

f1 ori

Kan/neo

pUC ori

pEGFP-eIF5A5200 bp

Plasmid name: pEGFP-eIF5A

80

51

28.6

35.9

122

20.8

anti-eIF-5A

anti-GFP

B

Transient transfection

Expression vector pEGFP-CMV

NdeI 1000enterokinase 982NcoI 955EcoRI 935

NdeI 483NruI 209

BsrGI/SspBI 1723XhoI 1745SacI 1748BamHI 1772

SmaI 2685

XhoI 3806

XbaI 3528SmaI 2685

BamHI 1772

CMV IEFLAG

HMK site

GFP

IVS

IRES

neoPoly A

Amp

pCINFG6120 bp

NdeI 1000enterokinase 982NcoI 955EcoRI 935

NdeI 483NruI 209

BsrGI/SspBI 1723XhoI 1745SacI 1748HindIII 1753

KpnI 2076

BamHI 2245

SmaI 3195

XhoI 4316

XbaI 4038

SmaI 3195

BamHI 2245

CMV IEFLAG

HMK site

GFP

eIF-5A

IVS

IRESneo

Poly A

Amp

pCINFGeIF-5A6630 bp

Stable transfection vector

IRES: internal ribosome entry sitePolyA: polyadenylation siteAmp: ampicilin resistant geneNeo: neomycin resistant geneCMV: cytomegalovirus promoter

Stable transfectants

FG

FG:5A_wt

FG:5A_K50R

GFP Phase contrast

Principle of regulated expression: In the expression system, the repressor (R) is synthesized under the control of an appropriate promoter (P). When the effector (E) is absent, the repressor will bind to one or more operator sites (with dyad symmetry)within a promoter/enhance region and inhibits the transcription initiation. In the presence of E, repression is relieved, thereby allowing transcription of gene X.

Inducible or Regulated ExpressionThe gene X is regulated and is expressed only under the induction condition.

Tetracycline-regulated expression system

Tetracycline: discovered in 1948, ~1000 derivatives by 1980, global production 500 metric tons; gaining entry via porin channels, binding to ribosomes and disrupting codon-anticodon interaction

TetR: repressor protein (helix-turn-helix), binds to tetO that controls tetRand tetA, tetracycline binding reduces the TetR-tetO binding

Resistant proteins are multimeric antiporter proteins called Tet proteins at inner membrane such as TetA (399 aa), exporting tetracycline from the cell, under control of tetO, TetA overexpression leads to bacterial death. TetA is controlled by tetR. tetA give bacteria tetracycline resistance.

Inducible or Regulated ExpressionThe gene to be expressed is regulated, expressed only under the induction condition.

• The Tet repression system: Target gene is controlled by tetO elements upstream. Tet repressor (TetR) represses the expression of the target gene and tetracycline releases the suppression.

• The Tet trans-activator system: A fusion protein tTA (TetR/VP16) activates tetO and thus the target gene expression. Tetracycline binding abolishes the activation

• The Tet reversed activator system: rtTA (mutated TetR/VP16) activates tetO in the presence of tetracycline

• The autoregulatory Tet system: The fusion regulator protein tTA is under control of tetO

Inducible or Regulated Expression

TetR repression system (TetR)Tet transactivation system (TetR/VP16 = tTA (+Tet inactivate)Tet-reversed activation system (rTetR+VP16 -> rTetR/VP16 (+Tet activate)

The Tet regulator pasmid: Regulator vector will be integrated into the genome of the host cell line which encodes tTA (tetracycline-controlled transactivator) or rtTA (reverse tTA). tTA is a hybrid protein made of TetR and the VP16 activation domain. pTet-On expresses rtTA, which activates tetO-driven transcription in the presence of doxycycline. pTet-Off expresses tTA, which activates transcription in the absence of doxycycline.

pTRE2 encodes the tetracycline response element (TRE), which contains seven repeats of tetO sequence. Target gene is inserted into MCS. Co-transfection of pTK-Hyg or other marker vector is required for stable transfectants selection

from pTet-Off

from pTet-On

Tet-off system

Ecdysone-inducible Gene Expression System

Regulator plasmid: VgEcR (truncated ecdysone receptor fused with VP16 activation domain) and retinoid X receptor

Response plasmid: 4 or 5 copies of E/GRE (hybrid ecdysone/glucocorticoid response elements),

Regulator plasmid

Response plasmid

Vector: pQEHost: E. coli M15 strainFusion tag: 6xHisInduction and repression system: lac operonPurification strategy: metal affinity chromatography

www.qiagen.com

QIAexpress System

Recombinant protein production: Plasmid design, cloning, transformation, bacteria growth, recombinant protein induction, cell lysis, protein extraction, metal affinity chromatography, gel electrophoresis

History• Porath et al (1975) Metal chelate affinity chromatography, a new approach to

protein fractionation. Nature 258, 598-599.• Sulkowski (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1-7.• Houchili et al (1987)New metal chelate adsorbent selective for proteins and

peptides containing neighboring histidine residues. J Chromatog. 411, 177-184.• Houchili (1989) Genetically designed affinity chromatography using a novel

metal chelate absorbent, Biologically active molecules 217-239.

pQE Strategy

pQE: Belong to the pDS family of plasmidsFeatures:

T5 promoter, 2 lac operator , Synthetic RBSII, 6xHis MCS with stop codon, Two transcriptional terminators β-Lactamase gene ColE1 origin of replication

E. coli host strain M15 [pREP4] contains pREP4 plasmid which confers kanamycinresistance and constitutively expresses the lacrepressor protein encoded by the lac 1 gene. The pREP4 plasmid is compatible with all plasmids carrying the ColE1 origin of replication.

Positions of elements in bases

Recombinant constructs based on different pQE vectors

Insert a 6xHis tag into an existing expression vector

pQE-TriSystem vector

pQE-TriSystem vector for parallel protein expression using a single construct in E. coli, insect, andmammlian cells. PT5: T5 promoter, lac O: lac operator, RBS: ribosome binding site, ATG: start codon,8xHis: His tag sequence, MCS: multiple cloning site, Stop Codons: stop codons in all three reading frames,Ampicillin: ampicillin resistance gene, P CAG: CMV/actin/globin promoter, P p10: p10 promoter,Kozak: Kozak consensus sequence, termination region: transcription terminator region,lef2, 603/1629: flanking baculovirus sequences to permit generation of recombinant baculoviruses,pUC: pUC origin of replication.

Competent cells

Mandel, M. and Higa, A. (1970) Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53, 54.

Cohen, S., Chang, A.C.Y. and Hsu, L. (1973) Nonchromosomal antibiotic resistance in bacteria: genetic transformation in E.coli by R-factor DNA. PNAS 69, 2110.

Transformation efficiency: >108 per µg DNA

Detection of positive expression clones by colony blotting

Purification schemePurification can be performed under native or denaturing conditions.

Inclusion bodies (nature of the protein, the host cell, level of expression, growth and induction conditions).

The expression level can range from <1 to 40%.

Purification under native condition: Human SRF expressed from a vacciniavirus vector in HeLa cells and purified using Ni-NTA agarose: 1, cell lysate; 2, flow-through; 3, 0.8 mMwash; 4&5, 8 mM wash; 6&7, 40 mM wash; 8&9, 80 mM elution.