cloning a gene for over-expression and puriﬁcation ... · pdf filecloning a gene for...

A.-F. Miller © 2013 Page

Cloning a Gene for Over-expression and Purification

Motivation: produce a bioremediation enzyme to metabolize herbicide residues in soil.

Nitroreductase is able to metabolize herbicides such as 2,4-dinitrophenol. However this enzyme denatures readily: is it too delicate for field work.

NADH-oxidase (NADOX) is a homologous enzyme from Thermus thermophilus that has a very high melting temperature and at 25° C displays increased catalytic activity in the presence of denaturants and high salt concentrations.

We want to over-express NADOX and test if for possible use as a bioremediation tool.

Strategy: clone the gene into an overexpression plasmid.

1 A.-F. Miller, 2013, pg

Cloning a geneA clone is a (population of) genetically identical organisms.

Plasmid, small extrachromosomal circle of DNA. (4.3 kB vs. 4.6 MB) Includes origin of replication. Often carries a set of genes needed for metabolism of occasional nutrient sources, or antibiotics.

In order for the gene to be carried over multiple generations, it needs to be replicated at least as often as the cell in which it is carried. For convenience, we clone bacterial genes into plasmids.

A.-F. Miller, 2013, pg

Simplest example

3

Cloning vectors contain an origin of replication, a selectable marker, cloning sites.Splicing a gene of interest into a plasmid provides for its propagation by host cells and permits its ready repurification (retrieval).

G&G Fig. 12.1 A.-F. Miller, 2013, pg

Cloning operations do not depend on the

content of the gene.

4

Restriction enzymesvector, insert.sticky ends / blunt endsPhosphatase, ligaseDirectional cloning behind a promotor,transformation of ‘competent’ cells, selection.

G&G Fig. 12.3, 5


Making lots of the insert: use PCR

5

PCR: Polymerase chain reaction is used to make many copies of DNA lying between the primers used.Primers are necessary in order for DNA polymerase to copy template DNA.

5’ to 3’

Template

Primer

Polymerase


PCR amplification

6

Supply template, primers (two) NTP, DNA polymerase that tolerates repeated heating (to separate DNA strands) and cooling (to permit primers to anneal onto template).

A.-F. Miller, 2013, pg 7

PCR see also Fig 12.21

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linkers bind

+NTPpolymerase

melt 95°Ccool 70°C

primers at x1000


PCR, the video

8 Also http://www.youtube.com/watch?v=x5yPkxCLads

A.-F. Miller, 2013, pg 9 http://www.wehi.edu.au/education/wehitv/

DNA Replication


! Restriction enzymes are enzymes that cut DNA at specific sequences within double stranded DNA.

! Different enzymes cut DNA at different sequences.! Target sequences are usually palindromic (read the same in both

directions).! These enzymes can be used to confirm the presence of sequences by

virtue of their action of cutting DNA only when the sequence occurs.! These enzymes often cut the two DNA strands in staggered locations,

and so are also used to produce complementary overhangs “sticky ends”. ! Sticky ends allow different digestion products to be assembled together

to produce recombinant DNA molecules from two pieces that have been produced by the same restriction enzymes.We will be using Nde I (CATATG) and Hind III (AAGCTT)

5‘CGTACCGATCATATGTACCGGAT3’3‘GCATGGCTAGTATACTGGAATA5’

5‘CGTACCGATCA3‘GCATGGCTAGTAT

+

TATGTACCGGAT3’ ACATGGAATA5’

Restriction endonucleases

TATGGCTTGGAAT3’ ACCGAACCTTA5’

5‘CGTACCGATCATATGGCTTGGAAT3’3‘GCATGGCTAGTAT ACCGAACCTTA5’

Nde I

10


gene of interest

pET plasmid provides a strong promotor and other ‘designer’ features in the service of expressing a gene of interest.

Insert gene here

We choose to use a DNA polymerase that has no other duties in the cell, and that we can up-regulate at will (induce) upon addition of IPTG to the medium.(Look up IPTG)

Over-expression vectors

11 A.-F. Miller © 2013 Page

SphI (725)*

BglII (528)*T7 forwardT7

XbaI (462)*NcoI (423)*

HisTEV

NdeI (377)*EcoRI (368)

BamHI (359)*SacI (354)*

AccI (344)XhoI (328)*

T7 reverseT7 term

EcoRV (192)NheI (32)*

AccI (3624)BsmBI (3495)

ApaI (1461)*

BssHII (1661)*EcoRV (1700)

BsmBI (1865)

pBR322 origin

BsaI (4808)*

PstI (4992)*AmpR

AatII (5669)*EcoRI (5740)

pET15TEV_NESG

5741 bp

Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III| | TEV Protease | | | | | | | CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC ______6XHis tag____

Vector: pET15TEV_NESG

rbs

Our Choice:

Provides N-terminal His tag,TEV cleavage siteribosome binding site,(but no lac repressor).

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Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144

lacI (640-1719)

ori (3153)

Ap (3

914-

4771

)

f1 origin (4903-5358)

Sty I(57)Bpu1102 I(80)

Ava I(158)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)

Bgl II(268)SgrA I(309)

Sph I(465)EcoN I(525)

PflM I(572)ApaB I(674)

Mlu I(990)Bcl I(1004)

BstE II(1171)Bmg I(1199)Apa I(1201)

BssH II(1401)EcoR V(1440)

Hpa I(1496)

PshA I(1835)

Psp5 II(2097)Bpu10 I(2197)

BspE I(2617)Tth111 I(2836)

Bst1107 I(2862)Sap I(2975)

BspLU11 I(3091)

AlwN I(3507)

Bsa I(4045)

Pst I(4229)

Pvu I(4354)

Sca I(4464)

Dra III(5127)

pET-21(+) (Cat. No. 69770-3) is a transcription vector designed for expression from bacterialtranslation signals carried within a cloned insert. It therefore lacks the ribosome binding site andATG start codon present on the pET translation vectors. A C-terminal His•Tag® sequence is avail-able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322convention, so the T7 expression region is reversed on the circular map. The cloning/expressionregion of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNAthat corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat. No. 69337-3).

pET-21(+) cloning/expression region

TB063 12/98

pET-21(+) sequence landmarks

T7 promoter 237-253T7 transcription start 236Multiple cloning sites(BamH I - Xho I) 158-203His•Tag® coding sequence 140-157T7 terminator 26-72lacI coding sequence 640-1719pBR322 origin 3153bla coding sequence 3914-4771f1 origin 4903-5358

pET-21(+) Vector

pET-21(+)(5369bp)Eam1105 I(3984)–

T7 terminator primer #69337-3

T7 promoter

T7 promoter primer #69348-3

EcoR I Xho I

T7 terminator

BamH I Sac I Sal I Hind III Not Ilac operator

Bpu1102 I

Ava I*

Sty I

Eag IHis•Tag


lacI (640-1719)

ori (3153)

Ap (3

914-

4771

)

f1 origin (4903-5358)

Sty I(57)Bpu1102 I(80)





Mlu I(990)Bcl I(1004)



Hpa I(1496)

PshA I(1835)

Psp5 II(2097)Bpu10 I(2197)

BspE I(2617)Tth111 I(2836)

Bst1107 I(2862)Sap I(2975)

BspLU11 I(3091)

AlwN I(3507)

Bsa I(4045)

Pst I(4229)

Pvu I(4354)

Sca I(4464)

Dra III(5127)



TB063 12/98



pET-21(+) Vector

pET-21(+)(5369bp)Eam1105 I(3984)–


T7 promoter


EcoR I Xho I

T7 terminator


Bpu1102 I

Ava I*

Sty I

Eag IHis•Tag

rbs geneATG TAA

Vector: pET21

Second Choice:

Provides lac operator repress expression of toxic gene, C-terminal His tag.


CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG

Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III | | TEV Protease | | | | | | |

Gene sequence TTHA0425NCBI Reference Sequence: NC_006461.1>gi|55979969:402495-403112 Thermus thermophilus HB8 chromosome, ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCGTTCCATCCGGCGTTACCGGAAGGACCCCGTACCCGAGGGGCTTCTCCGGGAAATCCTCGAGGCCGCCCTCCGGGCGCCCTCGGCCTGGAACCTCCAGCCCTGGCGGATCGTGGTGGTGCGGGACCCCGCCACCAAACGGGCCCTGAGGGAGGCGGCCTTCGGCCAGGCCCACGTGGAGGAGGCCCCCGTGGTCCTGGTCCTCTACGCCGACCTCGAGGACGCTCTCGCCCACCTGGACGAGGTCATCCACCCCGGGGTCCAGGGGGAAAGGCGTGAGGCGCAGAAGCAGGCCATCCAACGGGCCTTCGCCGCCATGGGGCAAGAGGCGCGAAAGGCCTGGGCCTCCGGGCAGAGCTACATCCTCTTGGGCTACCTCCTTCTCCTCCTGGAGGCTTATGGCCTCGGAAGCGTCCCCATGCTGGGGTTTGACCCCGAGAGGGTGAGGGCGATCCTGGGGCTTCCTTCCCACGCCGCCATCCCCGCCCTGGTGGCCTTGGGCTACCCGGCGGAGGAGGGCTACCCCTCCCACCGCCTGCCCCTGGAGCGGGTGGTCCTCTGGCGCTAA

NADOX gene

Cannot use Xho I

Before proposing to use Nde I, Eco RI, Hind III or Xho I,confirm that these do not cut within the gene.Search for CATATG, GAATTC, AAGCTT, CTCGAG

Eenie Meenie Minie Moe, which nuclease should we use ?

mcs of pET15TEV

Blue coloured codons are corrections I made

based on literature disagreements and the

crystal structure.14


Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III| | TEV Protease | | | | | | | CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC ______6XHis tag____

Vector: pET15TEV_NESG

Sites we will use

Therefore we will need the following flanking sequences on our primers

‘5-CATATG➝ ←TTCGAA-5’

Both of these endonucleases need some bases on either side in order to cut efficiently, so we will add a few more bases.

5’-TTTTTTCATATG➝

←TTCGAATTTTTT-5’15 A.-F. Miller © 2013 Page

Add a linker between protease cleavage and our protein.

ATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCG

5'3' Frame 1Met G H H H H H H E N L Y F Q S H Met A N S A D P A S S V D A K L L

http://web.expasy.org/translate/

Insert a mini-linker of flexible soluble amino acids.Gly-Ser-

Therefore add codons for Gly and Ser to gene.

GS

Intelligent choice of codons

http://muta-tion.blogspot.com/2011/07/genetic.html

What codons to use for Gly and Ser ?

Design feature for the insert (our gene)

16


Hershberg R, Petrov DA (2009) General Rules for Optimal Codon Choice. PLoS Genet 5(7): e1000556. doi:10.1371/journal.pgen.1000556

For Gly, use GGC


For Ser, AGC , also Welch et al (2009)

Welch M, Govindarajan S, Ness JE, Villalobos A, Gurney A, et al. (2009) Design Parameters to Control Synthetic Gene Expression in Escherichia coli. PLoS ONE 4(9): e7002. doi:10.1371/journal.pone.0007002

Arg

Leu

Ser

Ile

Note that just choosing the most common codon does not guarantee higher expression.

18


Modified early sequence of our gene.5’-TTTTTTCATATGGGCAGC➝

The authentic gene (early bases, corrections in blue, start in green)

ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCG

Meld together to get: (only first 30 bases to be replicated are shown)

5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝

How far into the gene does the primer need to extend ?Long enough that it binds selectively (short sequences can recur elsewhere in the

genome simply at random), short enough that the search for complementary sequence does not happen too slowly (fast binding and

release from wrong sites).

extension, restriction site, codons for linker


Hence the following “Rules of thumb” for primer design

5’ end of the gene

Restriction site permits splicing into vector. (we have the Nde I site)ATG encodes start of translation. ( √)follows a ribosome binding site and a transcription initiation site. (plasmid provides these).Extend the primer by 2-10 bases upstream of restriction site (√).Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.

Tm = 2°C * (A + T) + 4°C * (C + G)http://www.basic.northwestern.edu/biotools/oligocalc.html

See http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/

We will deal with these next.

20



Use tools on web sites to determine the GC content and melting temperatures (Tm) of duplexes predicted from different lengths of oligos.

Considering first round only when only black bases pair.

Range of Tm values account for range of Na+ concentrations that may be present (stabilizing duplex).

Considering later rounds when daughter molecules from early rounds are serving as templates.


49 % GC, Tm = 68 - 78 °C 39 bases

50 % GC, Tm = 67 - 74 °C 32 nucleotides But only 14 b long in round 1, 2, 64% GC, Tm 43-47 °C

50 % GC, Tm = 69 - 79 °C 40 nucleotides22 bases long in round 1, 2, 59% GC, Tm 59-67 °C.

Only 21 b in rounds 1,2, 57 % GC, Tm = 56 - 63 °C


5’ end of the gene

http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi

Might RNA structure mask the start site ?

√ Predicted stability of -6.2 kcal/mol (would like to have < 10).

CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGGCAGCGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG

Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III | | TEV Protease | | | | | | |

Vector

5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝

22


Reverse primer, Rules of thumb

3’ end of the geneRestriction site permits splicing into vector.TAA stop codon (TAG and TGA are more prone to read-through).Extend the primer by 2-10 bases upstream of restriction site.Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.

Check that the two primers do not pair with one another.

5’ CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAG 3’

5’ CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAA AAGCTT AAAAAA 3’

3’ AC GGG GAC CTC GCC CAC CAG GAG ACC GCG ATT TTCGAA TTTTTT 5’

5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG GGG CA 3’

Mutate to a better stop codon. Add Hind III site and

extension.The primer is the complement strand

The primer (written right way ‘round, with site of Hind III cut)


Reverse primer, Rules of thumb3’ end of the geneRestriction site permits splicing into vector. (√)TAA stop codon (TAG and TGA are more prone to read-through). (√)Extend the primer by 2-10 bases upstream of restriction site. (√)Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.

Check that the two primers do not pair with one another.

5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG G 3’

The primer (bases that pair in the first round are in bold, all pair in rounds >2)

50 % GC Tm 67-77 °C 36 bases long (23 bases in rounds 1,2, 70 % GC and Tm 64-72 °C)45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)

Use green primer and a few extra cycles24


Primers for use with pET15TEV_NESG

5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝

5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C➝ 3’

Forward

Reverse

Consequences for expression:At N-terminus, after TEV cleavage, we will add H M G S to the ‘front’ of the protein.At the C-terminus there will be no changes relative to the native protein.

Check to be sure that your two primers will not bind strongly to one-another.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

49 % GC, Tm = 68 - 78 °C 39 bases Only 21 b in rounds 1,2, 57 % GC, Tm = 56 - 63 °C

45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)

Temperatures match well.



lacI (640-1719)

ori (3153)

Ap (3

914-

4771

)

f1 origin (4903-5358)

Sty I(57)Bpu1102 I(80)





Mlu I(990)Bcl I(1004)



Hpa I(1496)

PshA I(1835)

Psp5 II(2097)Bpu10 I(2197)

BspE I(2617)Tth111 I(2836)

Bst1107 I(2862)Sap I(2975)

BspLU11 I(3091)

AlwN I(3507)

Bsa I(4045)

Pst I(4229)

Pvu I(4354)

Sca I(4464)

Dra III(5127)



TB063 12/98



pET-21(+) Vector

pET-21(+)(5369bp)Eam1105 I(3984)–


T7 promoter


EcoR I Xho I

T7 terminator


Bpu1102 I

Ava I*

Sty I

Eag IHis•Tag


lacI (640-1719)

ori (3153)

Ap (3

914-

4771

)

f1 origin (4903-5358)

Sty I(57)Bpu1102 I(80)





Mlu I(990)Bcl I(1004)



Hpa I(1496)

PshA I(1835)

Psp5 II(2097)Bpu10 I(2197)

BspE I(2617)Tth111 I(2836)

Bst1107 I(2862)Sap I(2975)

BspLU11 I(3091)

AlwN I(3507)

Bsa I(4045)

Pst I(4229)

Pvu I(4354)

Sca I(4464)

Dra III(5127)



TB063 12/98



pET-21(+) Vector

pET-21(+)(5369bp)Eam1105 I(3984)–


T7 promoter


EcoR I Xho I

T7 terminator


Bpu1102 I

Ava I*

Sty I

Eag IHis•Tag

rbs geneATG TAA

provide Eco RI, rbs and atg. (NdeI underlined)

GAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT TTG GAC G...

Forward primer

Reverse primer: as for pET15TEV

NEXT: pET21

26


provide Eco RI, rbs and atg. (NdeI underlined)

5’-TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT T...

Forward primer: (rules of thumb)

Restriction site permits splicing into vector. (we have the Eco RI site)ATG encodes start of translation. ( √)Follows a ribosome binding site and a transcription initiation site. (√).Extend the primer by 2-10 bases upstream of restriction site. Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.

42 bases 40% GC 66-76 °C Tm in later rounds; 17 bases 59% GC 50-55 °C Tm in rounds 1,241 bases 39% GC 65-75 °C Tm in later rounds; 16 bases 56% GC 46-51 °C Tm in rounds 1,2

Use blue primer45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2


Primers for use with pET21

5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C ➝3’

Forward

Reverse

Consequences for expression:At N-terminus, The native protein will be intact (no changes).At the C-terminus there will be no changes relative to the native protein.

Check to be sure that your two primers will not bind strongly to one-another.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

31 bases 45 % GC Tm 62-71 °C (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)

Temperatures match alright.

5’ TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GT➝ 45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2

28

cloning a gene for over-expression and puriﬁcation ... · pdf filecloning a gene for...

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