Agilent pET System Vector Instruction Manual PDF

pET System Vectors and Hosts

Instruction Manual

Catalog #211521, #211523, #211621, #211623 Revision C0

For Research Use Only. Not for use in diagnostic procedures. 211521-12

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pET System Vectors and Hosts

CONTENTS Materials Provided ...................................................................................................................... 4

Storage Conditions ...................................................................................................................... 4

Additional Materials Required .................................................................................................. 5

Academic and Nonprofit Laboratory Assurance Letter.......................................................... 5

Introduction ................................................................................................................................. 6

pET Expression Vectors ................................................................................................ 6

pET-3 Vector Map ......................................................................................................... 8

pET-11 Vector Map ....................................................................................................... 9

Bacterial Strains .......................................................................................................... 10

Bacteriophage CE6 ...................................................................................................... 11

Cloning Protocol ........................................................................................................................ 12

Preparing the Vectors .................................................................................................. 12

Ligating the Insert........................................................................................................ 13

Transformation Protocol .......................................................................................................... 14

Transformation of the BL21-Gold Expression Strains ................................................ 14

Transformation Summary for the pUC18 Control Plasmid ......................................... 15

Expression Protocols ................................................................................................................. 15

Induction of Target Protein Using IPTG ..................................................................... 15

Induction of Target Protein by Infection with Lambda CE6 ....................................... 16

Growth and Maintenance of High-Titer Bacteriophage Lambda CE6 Stocks ............ 17

Phage Amplification .................................................................................................... 17

Induction of Target Protein by Infection with Lambda CE6 ....................................... 17

Troubleshooting ........................................................................................................................ 19

Preparation of Media and Reagents ........................................................................................ 20

References .................................................................................................................................. 22

MSDS Information .................................................................................................................... 22

Quick-Reference Protocol ........................................................................................................ 23

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pET System* Vectors and Hosts

MATERIALS PROVIDED Kit Quantity Catalog # Storage

pET expression systems

pET 3 vector series: 3a, b, c and d DNAa,b Four 20-g tubes of cesium chloride-banded, supercoiled plasmid DNA

#211621 20C

BL21-Gold(DE3) competent cells 10 100 l 80C

BL21-Gold(DE3)pLysS competent cells

pUC18 control plasmid (0.1 ng/l in TE buffer)

10 100 l

2 10 l

80C

20C

pET 11 vector series: 11a, b, c and d DNAa,b,c Four 20-g tubes of cesium chloride-banded, supercoiled plasmid DNA

#211623 20C

BL21-Gold(DE3) competent cells 10 100 l 80C

BL21-Gold(DE3)pLysS competent cells

pUC18 control plasmid (0.1 ng/l in TE buffer)

10 100 l

2 10 l

80C

20C

pET vectors

pET 3 vector series: 3a, b, c and d DNAa,b Four 20-g tubes containing cesium chloride-banded, supercoiled plasmid DNA

#211521 20C

pET 11 vector series: 11a, b, c and d DNAa,b,c Four 20-g tubes containing cesium chloride-banded, supercoiled plasmid DNA

#211523 20C

a The pET 3a, b, c and pET 11a, b, c plasmids have one base pair shift in the BamH I site, from a to b and b to c. b The pET 3d and 11d plasmids have an Nco I cloning site, which is not present in a, b and c. c The pET 11 series includes the lac operator and lacI repressor gene for tighter expression control (not in the pET 3 series).

STORAGE CONDITIONS Vectors: 20C Competent Cells: 80C

* The pET system is covered by U.S. Patent No. 4,952,496. A nondistribution agreement accompanies the products. Commercial customers must obtain a license agreement from Associated Universities before purchase. Revision C0 Agilent Technologies, Inc. 2007-2017

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ADDITIONAL MATERIALS REQUIRED IPTG

-Mercaptoethanol and Falcon 2059 polypropylene tubes calf intestinal alkaline phosphatase T4 ligase

ACADEMIC AND NONPROFIT LABORATORY ASSURANCE LETTER The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and is protected by U.S. patents assigned to Brookhaven Science Associates (BSA). BSA will grant a nonexclusive license for use of this technology, including the enclosed materials, based on the following assurances: 1. These materials are to be used for noncommercial research purposes only. A separate license is required for any commercial use, including the use of these materials for research purposes or production purposes by any commercial entity. Information about commercial licenses may be obtained from the Office of Intellectual Property and Industrial Partnerships, Brookhaven National Laboratory, Bldg. 475D, Upton, New York, 11973 [telephone (631) 344-7134]. 2. No materials that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase, may be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of this license and agrees to be bound by its terms. This limitation applies to strains BL21-Gold(DE3) and BL21-Gold(DE3)pLysS included in this kit and any derivatives you may make of them. You may refuse this license by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this license.

Commercial Entities Outside of the US The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and is protected by U.S. Patents assigned to Brookhaven Science Associates (BSA). To protect its patent properties BSA requires commercial entities doing business in the United States, its Territories or Possessions to obtain a license to practice the technology. This applies for in-house research use of the T7 system as well as commercial manufacturing using the system. Commercial entities outside the U.S. that are doing business in the U.S., must also obtain a license in advance of purchasing T7 products. Commercial entities outside the U.S. that are using the T7 system solely for in-house research need not obtain a license if they do no business in the United States. However all customers, whether in the U.S. or outside the U.S. must agree to the terms and conditions in the Assurance Letter which accompanies the T7 products. Specifically, no materials that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase, may be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of the assurance letter and agrees to be bound by its terms. This limitation applies to strains BL21-Gold(DE3) and BL21-Gold(DE3)pLysS included in this kit and any derivatives you may make of them. To obtain information about licensing, please contact the Office of Intellectual Property and Industrial Partnerships, Brookhaven National Laboratory, Building 475D, Upton, NY 11973 [telephone: 631-344-7134; Fax: 631-344-3729].

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INTRODUCTION The pET expression system1 is one of the most widely used systems for the cloning and in vivo expression of recombinant proteins in E. coli. This is due to the high selectivity of the pET systems bacteriophage T7 RNA polymerase for its cognate promoter sequences, the high level of activity of the polymerase and the high translation efficiency mediated by the T7 gene 10 translation initiation signals. In the pET system, the protein coding sequence of interest is cloned downstream of the T7 promoter and gene 10 leader sequences, and then transformed into E. coli strains. Protein expression is achieved either by IPTG induction of a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter, or by infection with the polymerase-expressing bacteriophage lambda CE6.2 Due to the specificity of the T7 promoter, basal expression of cloned target genes is extremely low in strains lacking a source of T7 RNA polymerase. Upon induction the highly active polymerase essentially out-competes transcription by the host RNA polymerase. This phenomenon, together with high-efficiency translation, achieves expression levels in which the target protein may constitute the majority of the cellular proteinafter only a few hours.

pET Expression Vectors The pET expression vectors, derived from the pBR322 plasmid, are engineered to take advantage of the features of the T7 bacteriophage gene 10 that promote high-level transcription and translation. The bacteriophage- encoded RNA polymerase is highly specific for the T7 promoter sequences, which are rarely encountered in genomes other than T7 phage genome. First, this ensures that the T7 promoter will not be recognized by host cell RNA polymerase. Thus target genes are transcriptionally silent in the uninduced state--a feature that is very important if the gene to be expressed is toxic to the cell. Second, upon induction, the target gene is the only gene in the cell that will be transcribed by the highly active polymerase. In addition to the T7 promoter, all the vectors contain the gene 10 5 leader, which facilitates highly efficient translation. The protein coding sequence of interest may be cloned directly after the gene 10 initiation codon using the Nde I (pET-3,-11, a, b and c) or Nco I sites (pET-3d, and -11d). Alternatively, the pET-3 and pET-11 vectors contain BamH I cloning sites in all three reading frames relative to the gene 10 reading frame. Cloning the gene of interest using the BamH I site results in a fusion protein containing 13 N-terminal amino acids from gene 10. The gene 10 transcription terminator is also included downstream of the cloning sites to allow efficient termination of transcription, preventing transcriptional read-through of unwanted plasmid sequences and increasing the RNA polymerase density on the sequence of interest--allowing high level accumulation of the specific protein-coding RNA transcripts.

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Despite the strong selectivity of the T7 promoter for its phage-encoded polymerase, residual "leaky" expression of very toxic proteins from the basic pET-3 constructs can be lethal to the cell. To circumvent this problem and achieve more stringent control of expression, the lac operator has been inserted between the T7 promoter and translation initiation sequences in the pET-11 vectors, thereby allowing IPTG-mediated de-repression of the T7 promoter in addition to IPTG-induction of T7 polymerase from the lacUV5 promoter in the DE3 containing strains (see Bacterial Strains). In order to provide adequate levels of lacI protein to shut off T7 polymerase expression as well as T7 promoter transcription, the lacI gene is included on the pET-11 plasmids. All of the vectors in the pET-3 and pET-11 series contain the -lactamase gene for ampicillin resistance and the pBR322 origin of replication. Other features specific to the various vectors are listed in Table I below and in Figures 1 and 2. TABLE I Features of the pET System Vectors

Vector Promoter ATG cloning site

Reading frame

pET-3a T7 Nde I GGAa

pET-3b T7 Nde I GAT

pET-3c T7 Nde I ATC

pET-3d T7 Nco I ATC

pET-11a T7/lac O Nde I GGA

pET-11b T7/lac O Nde I GAT

pET-11c T7/lac O Nde I ATC

pET-11d T7/lac O Nco I ATC a Reading frame defined as the first full codon within the BamH I recognition sequence

(GGATCC) that is in the same frame as the gene 10 initiation codon.

8

pET-3 Vector Map

Feature

Nucleotide Position

pET-3a pET-3b pET-3c pET-3d

T7 promoter 119 119 119 119

ribosome binding site (RBS) 6672 6672 6672 6672

Nde I (pET-3ac) or Nco I (pET-3d) cloning site 7883 7883 7883 7883

T7 gene 10 translated leader 81113 81113 81113 80112

BamH I cloning site 117122 116121 115120 114119

T7 terminator 191237 190236 189235 188234

ampicillin resistance (bla) ORF 8401697 8391696 8381695 8371694

pBR322 origin of replication 18482515 18472514 18462513 18452512

FIGURE 1 The pET-3 vectors

P T7 BamH I

Nde I OR Nco I

T T7

ampicillin

pBR322 ori

RBS gene 10 leader

pET-3 vectors 4.6 kb

pET-3a3d Cloning Site Regions sequence shown (66122)

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC GGA TCC

BamH INde I

M A S M T G G Q Q M G R G S START

T7 gene 10 leader peptide

pET-3a

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CCG

BamH INde I

M A S M T G G Q Q M G R D P START

T7 gene 10 leader peptide

pET-3b

RBS

GAAGGAGATATACC ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG C

Nco I BamH I

M A S M T G G Q Q M G R I R START

T7 gene 10 leader peptide

pET-3d

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG

Nde I BamH I

M A S M T G G Q Q M G R I R START

T7 gene 10 leader peptide

pET-3c

RBS

9

pET-11 Vector Map

Feature

Nucleotide Position

pET-11a pET-11b pET-11c pET-11d

T7 promoter with lac operator 143 143 143 143

ribosome binding site (RBS) 7480 7480 7480 7480

Nde I (pET-11ac) or Nco I (pET-11d) cloning site 8691 8691 8691 8691

T7 gene 10 translated leader 89121 89121 89121 88120

BamH I cloning site 125130 124129 123128 122127

T7 terminator 199245 198244 197243 196242

ampicillin resistance (bla) ORF 6571514 6561513 6551512 6541511

pBR322 origin of replication 16652332 16642331 16632330 16622329

lacI repressor ORF 42095288 42085287 42075286 42065285

FIGURE 2 The pET-11 vectors

pET-11a11d Cloning Site Regions sequence shown (74130)

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC GGA TCC

BamH INde I

M A S M T G G Q Q M G R G S START

T7 gene 10 leader peptide

pET-11a

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CCG

BamH INde I

M A S M T G G Q Q M G R D P START

T7 gene 10 leader peptide

pET-11b

RBS

GAAGGAGATATACC ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG C

Nco I BamH I

M A S M T G G Q Q M G R I R START

T7 gene 10 leader peptide

pET-11d

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG

Nde I BamH I

M A S M T G G Q Q M G R I R START

T7 gene 10 leader peptide

pET-11c

RBS

Nhe I

Nhe I

Nhe I

Nhe I

lacIq

P T7/lac O BamH I

Nde I OR Nco I

T T7

ampicillin

pBR322 ori

RBS gene 10 leader

pET-11 vectors 5.7 kb

10

Bacterial Strains The BL21-Gold expression strains provided with catalog #211621 and #211623 are derivatives of the E. coli B strain BL21 (F- ompT r-B m-B). The BL21 strain is generally good for protein expression due to its deficiency in lon protease3 as well as the ompT outer membrane protease that can degrade proteins during purification. (For more information, see Table II Features of the BL21-Derived Expression Strains.) BL21 strains are rifampicin sensitive, allowing use of the drug to inhibit transcription of host cell polymerase in instances where background synthesis is undesirable. The strain BL21-Gold (DE3) [F ompT hsdS(rB

mB

) dcm+ Tetr gal (DE3) endA Hte] carries a lambda DE3 lysogen that has the phage 21 immunity region, the lacI gene and the lacUV5-driven T7 RNA polymerase expression cassette. Upon induction with IPTG, the lacUV5 promoter is de-repressed allowing over-expression of T7 RNA polymerase and thus expression of the T7-promoted target gene from the pET expression plasmid. In order to further reduce basal activity of T7 RNA polymerase in the uninduced state, the strain BL21-Gold(DE3) [F ompT hsdS(rB

mB

) dcm+ Tetr gal (DE3) endA Hte (pLysS Camr)] carries a derivative of the plasmid pACYC184, which expresses the T7 lysozyme gene at low levels. T7 lysozyme binds to T7 RNA polymerase and inhibits transcription by this enzyme. Upon IPTG induction, overproduction of the T7 RNA polymerase renders low level inhibition by T7 lysozyme virtually ineffective. In addition to inactivation of T7 RNA polymerase transcription, T7 lysozyme has a second function involving specific cleavage of the peptidoglycan layer of the E. coli outer wall. The inability of T7 lysozyme to pass through the bacterial inner membrane restricts the protein to the cytoplasm, allowing E. coli to tolerate expression of the lysozyme protein. This second function of lysozyme does, however, confer the advantage of allowing cell lysis under mild conditions. Cells expressing T7 lysozyme are subject to lysis under conditions that would normally only disrupt the inner membrane (e.g., freezethaw cycles or the addition of chloroform or a mild detergent such as 0.1% Triton X-100) due to the action of the protein on the outer wall when the inner membrane is disrupted. The BL21-Gold expression strains incorporate major improvements over the original BL21 strain. The BL21-Gold strains feature the Hte phenotype present in our highest efficiency strain, XL10-Gold ultracompetent cells.4 The presence of the Hte phenotype increases the transformation efficiency of the BL21-Gold cells to >1 108 cfu/g of pUC18 DNA. In addition, the gene that encodes endonuclease I (endA), which rapidly degrades plasmid DNA isolated by most miniprep procedures, is inactivated. These two improvements allow direct cloning of many protein expression constructs.

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In cases where the protein of interest is extremely toxic to E. coli or where clones obtained from direct transformation of BL21-Gold strains consistently show sequence anomalies, perform the initial cloning steps in cells in which the T7 promoter is inactive and that are RecA-(e.g. XL1-Blue competent cells). Strains carrying the lacIq gene may be useful for further inhibiting basal expression from the pET-11 vectors during subcloning.

TABLE II

Features of the BL21-Derived Expression Strains5

Expression Strain Induction Advantages Disadvantages

BL21-Gold (DE3) competent cells

IPTG induction of T7 polymerase from lacUV5 promoter

High-level expression;

Ease of induction, direct cloning in expression strain

Leaky expression of T7 polymerase can lead to uninduced expression of potentially toxic proteins.

BL21-Gold (DE3)pLysS competent cells

IPTG induction of T7 Ease of induction, reduced uninduced expression, direct cloning in expression strain

Slight inhibition of induced expression when compared with BL21-Gold (DE3).

BL21 competent cells Infection with lambda bacteriophage CE6

Tightest control of uninduced expression

Induction not as efficient as DE3 derivatives. Induction (infection) process is more cumbersome.

Bacteriophage CE6 In cases where target genes are too toxic to allow plasmids to be established in DE3 lysogens, T7 RNA polymerase can be delivered to the cell by infection with the bacteriophage CE6. By this method, no T7 RNA polymerase is present in the cell until the desired time of induction. CE6 expresses T7 RNA polymerase from the lambda pL and pI promoters, and carries the Sam7 lysis mutations. This bacteriophage will allow effective expression of target genes in BL21 cells, and presumably other nonrestricting hosts which absorb lambda. The phage can be propagated in the host strain LE392 [F-e14- (mcrA) hsdR514 (rk-mk+) supE44 supF58],6 which suppresses the Sam7 mutation and therefore allows lysis of infected cells.

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CLONING PROTOCOL

Preparing the Vectors

Perform a complete DNA digestion with the appropriate enzymes. (Perform Nde IBamH I or Nco IBamH I double digests to clone the gene of interest at the initiation codon. Perform a BamH I single digest to produce a fusion of the protein of interest to the gene 10 translated leader.) If the inserts to be cloned into these vectors contain one or more internal Nde I, Nco I or BamH I sites, PCR primers may be engineered to include restriction sites with overhangs compatible with the overhangs produced by the chosen enzymes.

Dephosphorylate the digested pET vector with calf intestinal alkaline phosphatase (CIAP) prior to ligating to the insert DNA. If more than one restriction enzyme is used, the background can be reduced further by electrophoresing the digested vector DNA on an agarose gel and gel purifying the desired vector band, leaving behind the small fragment excised from between the two restriction enzyme sites.

After gel purification, resuspend the vector DNA in a volume of TE buffer (see Preparation of Media and Reagents) that will allow the concentration of the vector DNA to be the same as the concentration of the insert DNA (~0.1g/l).

13

Ligating the Insert For ligation, the ideal insert-to-vector ratio of DNA is variable; however, a reasonable starting point is 2:1 (insert-to-vector molar ratio), measured in available picomole ends. This is calculated as follows:

600 pairs base ofnumber

10 2 DNAof gramends/micro Picomole

6

1. Prepare three control and two experimental 10-l ligation reactions by adding the following components to separate sterile 1.5-ml microcentrifuge tubes:

Note For blunt-end ligation, reduce the rATP to 0.5 mM and incubate the reactions overnight at 1214C.

Control Experimental

Ligation reaction components 1a 2b 3c 4d 5d

Prepared vector (0.1 g/l) 1.0 l 1.0 l 0.0 l 1.0 l 1.0 l

Prepared insert (0.1 g/l) 0.0 l 0.0 l 1.0 l X l X l

rATP [10 mM (pH 7.0)] 1.0 l 1.0 l 1.0 l 1.0 l 1.0 l

Ligase buffer (10)e 1.0 l 1.0 l 1.0 l 1.0 l 1.0 l

T4 DNA ligase (4 U/l) 0.5 l 0.0 l 0.5 l 0.5 l 0.5 l

Double-distilled (ddH2O) to 10 l 6.5 l 7.0 l 6.5 l X l X l a This control tests for the effectiveness of the digestion and the CIAP treatment. Expect a low number of transformant colonies if the digestion and CIAP treatment are effective. b This control indicates whether the vector is cleaved completely or whether residual uncut vector remains. Expect an absence of transformant colonies if the digestion is complete. c This control verifies that the insert is not contaminated with the original vector. Expect an absence of transformant colonies if the insert is pure. d These experimental ligation reactions vary the insert-to-vector ratio. Expect a majority of the transformant colonies to represent recombinants. e See Preparation of Media and Reagents.

2. Incubate the reactions for 2 hours at room temperature (22C) or overnight at 4C.

3. Transform 12l of the ligation mix into the appropriate competent bacteria. Plate on selective media.

Note For most applications, the ligation reaction may be transformed directly into BL21-Gold(DE3) or BL21-Gold(DE3)pLysS competent cells (provided with catalog #211621 and #211623). See Transformation of the BL21-Gold Expression Strains for a transformation protocol.

4. Verify the presence and orientation of the insert by PCR, restriction analysis or other appropriate methods.

14

TRANSFORMATION PROTOCOL

Transformation of the BL21-Gold Expression Strains

1. Thaw the competent cells on ice.

Note Store the competent cells on ice at all times while aliquoting. It is essential that the Falcon 2059 polypropylene tubes are placed on ice before the competent cells are thawed and that 100 l of competent cells are aliquoted directly into each prechilled polypropylene tube. Do not pass the frozen competent cells through more than one freezethaw cycle.

2. Gently mix the competent cells. Aliquot 100 l of the competent cells into the appropriate number of prechilled 15-ml Falcon 2059 polypropylene tubes.

3. Add 150 ng of DNA to each transformation reaction and swirl gently. For the control transformation reaction, add 1 l of the pUC18 control plasmid (provided with Catalog #211621 and #211623) to a separate 100-l aliquot of the competent cells and swirl gently.

4. Incubate the reactions on ice for 30 minutes.

5. Preheat SOC medium in a 42C water bath for use in step 8.

6. Heat-pulse each transformation reaction in a 42C water bath for 20 seconds. The duration of the heat pulse is critical for optimal transformation efficiencies.

7. Incubate the reactions on ice for 2 minutes.

8. Add 0.9 ml of 42C SOC medium to each transformation reaction and incubate the reactions at 37C for 1 hour with shaking at 225250 rpm.

9. Concentrate the cells from the experimental transformation by centrifugation (200 g for 35 minutes) and plate the entire transformation reaction (using a sterile spreader)ll onto a single LBampicillin agar plate.

To plate the cells transformed with the pUC18 control plasmid, first place a 195-l pool of SOC medium on an LBampicillin agar plate. Add 5 l of the control transformation reaction to the pool of SOC medium. Use a sterile spreader to spread the mixture.

10. Incubate the plates overnight at 37C. See Preparation of Media and Reagents. ll When spreading bacteria onto the plate, tilt and tap the spreader to remove the last drop of

cells. If plating <100 l of the transformation reaction, plate the cells in a 200-l pool of SOC medium. If plating 100 l, the cells can be spread directly onto the plates.

15

Transformation Summary for the pUC18 Control Plasmid Host strain

Plating quantity

Expected number of colonies

Efficiency (cfu/g of pUC18 DNA)

BL21-Gold(DE3) competent cells

5 l >50 1 108

BL21-Gold(DE3)pLysS competent cells

5 l >50 1 108

a The efficiencies quoted for the BL21-Gold(DE3) and the BL21-Gold(DE3)pLysS host strain competent cells (provided with Catalog #211621 and #211623) are guaranteed only if the user follows the storage and transformation protocols outlined in this manual. Efficiencies of competent cells prepared by the user cannot be guaranteed.

EXPRESSION PROTOCOLS

Induction of Target Protein Using IPTG The following induction protocol is a general guide for expression of genes under the control of IPTG-inducible promoters on an analytical scale (1 ml of induced culture). Most commonly, this protocol is used to analyze protein expression of individual transformants when using BL21-Gold(DE3) host strains in combination with plasmids containing T7 promoter constructs (e.g. pET vectors). Expression cassettes under the control of the trp/lac hybrid promoter, tac, can be also induced using this protocol. In the case of tac promoter constructs, non-DE3 lysogen strains can be employed as hosts.

Note The transformation procedure described above will produce varying numbers of colonies depending on the efficiency of transformation obtained using the expression plasmid. It is prudent to test more than one colony as colony-to-colony variations in protein expression are possible.

1. Inoculate 1-ml aliquots of LB broth containing 100 g/ml of carbenicillin or ampicillin with single colonies from the transformation. Shake at 220250 rpm at 37C overnight.

Note If the transformed cells contain a pACYC-based plasmid (e.g., the BL21-Gold(DE3)pLysS strain or any BL21-CodonPlus strain), the overnight culture must include chloramphenicol at a final concentration of 50 g/ml in addition to the carbenicillin/ampicillin required to maintain the pET plasmid.

2. The next morning, pipet 50 l of each culture into fresh 1-ml aliquots of LB broth containing no selection antibiotics. Incubate these cultures with shaking at 220250 rpm at 37C for 2 hours.

See Preparation of Media and Reagents.

16

3. Pipet 100 l of each of the cultures into clean microcentrifuge tubes and place the tubes on ice until needed for gel analysis. These will serve as the non-induced control samples.

4. To the rest of the culture in each tube add IPTG to a final concentration of 1 mM. Incubate with shaking at 220250 rpm at 37C for 2 hours.

Note These values for IPTG concentration and induction time are starting values only and may require optimization depending on the gene expressed.

5. After the end of the induction period, place the cultures on ice.

6. Pipet 20 l of each of the induced cultures into clean microcentrifuge tubes. Add 20 l of 2 SDS gel sample buffer to each.

7. Mix the tubes containing the non-induced samples to resuspend the cells and pipet 20 l from each tube into clean microcentrifuge tubes. Add 20 l of 2 SDS gel sample buffer to each.

8. Heat all tubes to 95C for 5 minutes and analyze the samples by Coomassie Brilliant Blue staining of an SDS-PAGE gel, placing associated non-induced/induced samples in adjacent lanes.

Induction of Target Protein by Infection with Lambda CE6 Expression of genes under the control of the T7 promoter (e.g. genes in pET vectors) can be achieved in non-DE3 lysogen host strains (e.g. BL21) if the strain harboring the expression plasmid is subsequently infected with lambda CE6. Lambda CE6 expresses T7 polymerase, which in turn drives the transcription of the gene downstream of the T7 promoter. The following protocols describe the growth and maintenance of lambda CE6 and the use of lambda CE6 for infecting host strains. We offer the Lambda CE6 Induction Kit (Catalog# 235200) for use in protein expression protocols which incorporate CE6 infection. See Preparation of Media and Reagents.

17

Growth and Maintenance of High-Titer Bacteriophage Lambda CE6 Stocks

1. Inoculate 5 ml of modified* NZY broth with a single colony of LE392 host cells. Shake overnight at 37C at 220250 rpm.

2. Centrifuge the overnight culture for 15 minutes at 17002000 g at 4C. Resuspend the cells in 10 mM MgSO4 to a final OD600 of 0.5.

3. Combine 250 l of cells (at OD600 = 0.5) with 1 106 pfu of CE6 in Falcon 2059 polypropylene tubes in triplicate. Incubate at 37C for 15 minutes.

4. Add 3 ml of melted NZY top agar (equilibrated to 48C) to each cell suspension and plate on warm agarose plates. Incubate the plates overnight at 37C.

5. Flood each plate with 5 ml of SM solution and rock the plates for 2 hours at room temperature.

6. Remove the SM solution (which contains the lambda CE6) from each plate and pool the volumes in a 50-ml conical tube.

7. Centrifuge the SM solution at 17002000 g for 15 minutes at 4C.

8. Remove the supernatant and determine the titer of the solution.

9. Store the lambda CE6 stock at 4C.

Phage Amplification If the titer drops over time, or if more phage are needed, grow up LE392 cells in 10 ml of medium and add bacteriophage lambda CE6 at a multiplicity of infection of 1:1000 (CE6-to-cell ratio). Continue growing the culture at 37C for 56 hours and spin down the cellular debris. Titer of the supernatant should be 5.0 109 pfu/ml. For general information regarding phage amplification, see reference 7.

Induction of Target Protein by Infection with Lambda CE6

Note This protocol is designed for inductions in 50-ml culture volumes. If inductions of larger volumes of culture are desired, it will be necessary to increase the volume of the overnight culture in step 1. The increased volume of overnight culture is necessary to achieve the required cell density (A600 1) in the larger volume of broth the following day.

* NZY broth to be used for lambda infection protocols should be supplemented with maltose

at a final concentration of 0.2%. Add 1 ml of 20% maltose solution (filter-sterilized) per 100 ml of NZY broth to achieve the correct final concentration of maltose.

See Preparation of Media and Reagents.

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1. Inoculate 5 ml of modified* NZY broth containing 100 g/ml of carbenicillin or ampicillin with a single colony of BL21 cells (not a DE3 lysogen) harboring the expression plasmid. Shake overnight at 37C at 200250 rpm.

Note If the host cells contain a pACYC-based plasmid (e.g., any BL21-CodonPlus strain), the overnight culture must include chloramphenicol at a final concentration of 50g/ml in addition to the carbenicillin/ampicillin required to maintain the pET plasmid.

2. In the morning, centrifuge 1.0 ml of the overnight culture, resuspend the cells in 1.0 ml of fresh modified* NZY broth, and pipet the resuspended cells into a flask containing 50 ml of fresh modified* NZY broth (no selection antibiotics).

3. Record the A600 of the diluted culture. It should be 0.1. If the A600 is > 0.1, use more fresh modified* NZY broth to dilute the culture to A600 0.1. If the A600 is < 0.1, the time required to reach an A600 of 0.3 (in step 4) will be extended.

4. Grow the culture to an A600 of 0.3 and add glucose to a final concentration of 4 mg/ml (e.g. 1.0 ml of a 20% glucose solution to the 50-ml culture).

5. Grow the culture to an A600 of 0.61.0 and add MgSO4 to a final concentration of 10 mM (e.g. 500 l of a 1.0 M solution of MgSO4 to the 50-ml culture).

6. Remove a portion of the culture to serve as the uninduced control and infect the rest with bacteriophage lambda CE6 at a multiplicity of infection (MOI) of 510 particles per cell. (To optimize induction, cultures may be split into 3 or 4 aliquots and infected with varying dilutions of bacteriophage lambda CE6. The subsequent induction can be monitored by SDS-PAGE or by a functional assay, if available.)

7. Grow the culture for 23 hours.

8. Remove 520 l of the culture for determination by SDS-PAGE, and harvest the remaining culture by centrifugation. Store the pellets at 70C.

Note If induction will be monitored using Coomassie stain, silver stain, or another nonspecific protein stain, include a control of CE6-infected BL21 cells harboring the plasmid without a cloned insert.

* NZY broth to be used for lambda infection protocols should be supplemented with maltose at a final concentration of 0.2%. Add 1 ml of 20% maltose solution (filter-sterilized) per 100 ml of NZY broth to achieve the correct final concentration of maltose.

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TROUBLESHOOTING Observation Suggestions

Poor induction Poor induction can occur for a number of reasons, including loss of plasmid due to instability resulting from expression of toxic proteins, unstable mRNA or poorly translated RNA that is high in secondary structure, and high abundance of codons which are rare in E. coli. BL21-CodonPlus Competent Cells are engineered to contain extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous proteins in E. coli.

Plasmid instabilityPrior to induction of cultures, determine the fraction of cells containing inducible plasmid by plating a 105fold dilution of cells on plates that have a) IPTG plus ampicillin, b) IPTG alone, and cells at a dilution of 2 106fold which have c) ampicillin or d) no additives Reduced colony formation should occur on ampicillin-containing plates relative to the LB plates. Successful induction of target gene on IPTG-containing plates should result in virtually no increase in bacterial growth; therefore, 2% of the cells should form colonies on plates that contain IPTG alone and 0.01% on plates that contain IPTG plus antibiotic. Conversely, cells that contain mutant plasmids that are poorly induced should show higher colony formation on the IPTG plates.

More tightly controlled induction may be desirable. Available methods include using a pET-11 vector (instead of a pET-3 vector) containing a lac operator; using BL21-Gold(DE3)pLysS (instead of BL21-Gold(DE3)); and, in extreme cases, performing induction by infection with the bacteriophage CE6. Also see Table II, Features of the BL21-Derived Expression Strains.

Problems associated with induction time

In certain cases accumulation of target protein may kill cells at saturation while allowing normal growth in logarithmically growing cultures, while in other cases target protein may continue to accumulate in cells well beyond the recommended 3-hour induction period. To determine the optimal time of induction, a time course may be carried out during which a small portion of the culture is analyzed by SDS-PAGE at various times following induction.

Inclusion bodiesIn some cases protein may form insoluble inclusion bodies at 37C. In many cases, this protein may be soluble and active if the induction is carried out at 30C. Inclusion body formation may be used as a purification step by simply spinning out the insoluble material from crude lysates and redissolving the protein in urea or guanidinium-HCl.

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PREPARATION OF MEDIA AND REAGENTS

LB Broth (per Liter) 10 g of NaCl 10 g of tryptone 5 g of yeast extract Adjust to pH 7.0 with 5 N NaOH Add deionized H2O to a final volume of

1 liter Autoclave

LB Agar (per Liter) 10 g of NaCl 10 g of tryptone 5 g of yeast extract 20 g of agar Adjust pH to 7.0 with 5 N NaOH Add deionized H2O to a final volume of

1 liter Autoclave Pour into petri dishes (~25 ml/100-mm

plate) LBAmpicillin Broth (per Liter)

1 liter of LB broth, autoclaved Cool to 55C Add 10 ml of 10-mg/ml filter-sterilized

ampicillin

LBAmpicillin Agar (per Liter) 1 liter of LB agar, autoclaved Cool to 55C Add 10 ml of 10-mg/ml filter-sterilized

ampicillin Pour into petri dishes

(~25 ml/100-mm plate)

LBAmpicillinMethicillin Agar (per Liter)

(Use for reduced satellite colony formation) 1 liter of LB agar Autoclave Cool to 55C Add 20 mg of filter-sterilized ampicillin Add 80 mg of filter-sterilized methicillin Pour into petri dishes (~25 ml/100-mm

plate)

NZY broth, (per Liter) 5 g of NaCl 2 g of MgSO4 . 7H2O 5 g of yeast extract 10 g of NZ amine (casein hydrolysate) Adjust the pH to 7.5 with NaOH

TE Buffer 10 mM Tris-HCl (pH 7.5) 1 mM EDTA

SOB Medium (per Liter) 20.0 g of tryptone 5.0 g of yeast extract 0.5 g of NaCl Autoclave Add 10 ml of 1 M MgCl2 and 10 ml of 1 M MgSO4/liter of SOB medium prior

to use Filter sterilize

SOC Medium (per 100 ml) SOB medium Add 1 ml of a 2 M filter-sterilized glucose

solution or 2 ml of 20% (w/v) glucose prior to use

Filter sterilize

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10 Ligase Buffer 500 mM Tris-HCl (pH 7.5) 70 mM MgCl2 10 mM dithiothreitol (DTT)

Note rATP is added separately in the ligation reaction.

Agarose Plates (per Liter) Melt 20 g of agarose in 500 ml of deionized

H2O Add the following: 5 g of NaCl 5 g of yeast extract 10 g of tryptone Add deionized H2O to a final volume of

1 liter Autoclave Pour into petri dishes (~25 ml/100-mm plate)

NZY Top Agar (per Liter) Prepare 1 liter of NZY broth Add 0.7% (w/v) agarose Autoclave

2 SDS gel sample buffer 100 mM Tris-HCl (pH 6.5) 4% SDS (electrophoresis grade) 0.2% bromophenol blue 20% glycerol

Note Add dithiothreitol to a final concentration in the 2 buffer of 200 mM prior to use. This sample buffer is useful for denaturing, discontinuous acrylamide gel systems only.

SM Solution 5 g of NaCl 2 g of MgSO4 . 7H2O 50 ml of 1 M Tris-HCl (pH 7.5) 5 ml 2% gelatin Add deionized H2O to a final volume of

1 liter Adjust the pH to 7.5 Autoclave

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REFERENCES 1. Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990) Methods

Enzymol 185:6089. 2. Studier, F. W. and Moffatt, B. A. (1986) J Mol Biol 189(1):11330. 3. Phillips, T. A., VanBogelen, R. A. and Neidhardt, F. C. (1984) J Bacteriol 159(1):283

7. 4. Jerpseth, B., Callahan, M. and Greener, A. (1997) Strategies 10(2):3738. 5. Weiner, M. P., Anderson, C., Jerpseth, B., Wells, S., Johnson-Browne, B. et al. (1994)

Strategies 7(2):4143. 6. Borck, K., Beggs, J. D., Brammar, W. J., Hopkins, A. S. and Murray, N. E. (1976) Mol

Gen Genet 146(2):199207. 7. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory

Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

MSDS INFORMATION Material Safety Data Sheets (MSDSs) are provided online at http://www.chem.agilent.com/en- US/search/library/Pages/MSDSSearch.aspx. MSDS documents are not included with product shipments.

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pET System Vectors and Hosts Catalog #211521, #211523, #211621, and #211623

QUICK-REFERENCE PROTOCOL

Prepare DNA insert containing the target gene

Prepare pET vector by digestion with the appropriate restriction enzyme(s), CIAP treatment and agarose gel purification

Ligate the prepared vector and insert DNA

Transform cloning host competent cells (e.g. Agilent XL1-Blue cells) and verify construct

Expression in E. coli

Transform BL21 expression host competent cells

Induce protein expression by IPTG addition or CE6 infection

Harvest cells; analyze cell pellets for target protein production

Purify target protein