T7 High Yield In vitro Translation

Materials

• S30 T7 High Yield In vitro Protein Expression from Promega

• DNase- and RNase-free 1.5ml microcentrifuge tubes

• plasmid DNA encoding the protein of interest

• floor incubator shaker or thermomixer

1. Remove the reagents from the freezer, and allow them to thaw on ice or in an ice-water bath.

2. Set up the following reactions on ice in a DNase- and RNase-free 1.5ml microcentrifuge tube:

Mix thoroughly by pipetting several times or vortexing gently, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube.

3. Mix thoroughly by pipetting several times or vortexing gently, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube

4. Quickly bring reaction to 37°C, and incubate the reactions with vigorous shaking for 1 hour (see Notes 12 and 13).

5. Stop the reaction by placing the tubes in an ice-water bath for 5 minutes.

6. Analyze the results of the reaction. See Section 6 for gel analysis of proteins. To evaluate the results of the positive control reaction, see Section 7.

Notes:

1. The reaction is very sensitive to nuclease contamination. Wear gloves, and use RNase- and DNase-free reagents and reaction tubes, and filtered pipette tips.

2. Higher protein expression levels can be achieved by increasing the amount of T7 S30 Extract, Circular, in the reaction (up to 45% of the reaction volume) while keeping the total volume constant. However, increasing the volume of the extract will reduce the total number of reactions.

Component	Standard Reaction	Negative Control (see Note 10)	Positive Control (see Note 11)
DNA template (see Notes 7–9)	1µg 2016, 2060	0	2µl
S30 Premix Plus (mix well prior to use)	20µl	20µl	20µl
T7 S30 Extract, Circular (mix gently prior to use)	18µl	18µl	18µl
Nuclease-Free Water to a final volume of	50µl	50µl	50µl

2060 pET30b-Tat-Flp-ecoli-6H , E coli Flp recombinase, His tag is at C terminus, no His tag at N terminus

MGRKKRRQRRRPPAGTSVSLKKKRKVSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRILEHHHHHH

2106 pET28b-iFLP

MGHHHHHHGMGAAGRKKRRQRRRPPAGTSVSLKKKRKVSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI

3. Template DNA and water purity are critical. If protein synthesis efficiencies are low, examine the quality of the template DNA and water. Read the discussion concerning DNA templates in Section 3.A.

4. If necessary, transcription by the endogenous E. coli RNA polymerase can be inhibited by the antibiotic rifampicin, while transcription by the phage T7 RNA polymerase is unaffected. To inhibit the endogenous RNA polymerase, add 1µl of 50µg/ml of rifampicin in water (Section 10.A) prior to adding the DNA template to the reaction. Adding excess rifampicin is unnecessary and may decrease protein synthesis levels.

5. To obtain fluorescently labeled or biotinylated protein, use 2µl of FluoroTect™ or Transcend™ tRNA in 50µl of reaction. Titrate the tRNAs if necessary. For incorporation of radioactive amino acids, we recommend using the E. coli T7 S30 Extract System for Circular DNA (Cat.# L1130).

6. The reaction volume can be scaled down to as little as 5µl without reducing protein yields. For reactions less than 25µl, we recommend assembling the reactions in PCR strip tubes or 96-well PCR plates; for reactions of 25–50µl, we recommend using 0.5ml microcentrifuge tubes; for reactions of 50–250µl, we recommend using 1.5ml microcentrifuge tubes.

7. The amount of DNA required might be template-dependent. We recommend 0.5–1µg/50µl reaction of <5kb plasmid DNA with a T7 promoter. Higher DNA concentrations (such as 2µg/50µl reaction) can be used for large plasmids and vectors with an E. coli promoter. If necessary, optimize the amount of DNA added. In general, reactions should not contain more than 4µg of DNA. An increased amount of DNA can increase the incidence of internal translational initiation or the number of prematurely arrested translation products. 

8. Refer to Section 3.A for a discussion on suitable DNA templates for this system.

9. The T7 S30 Extract, Circular, contains nuclease activity, preventing the use of linear DNA templates, such as PCR products. PCR products containing a T7 promoter and a ribosome binding site can be expressed using TnT® T7 Quick for PCR DNA (Cat.# L5540).

10. For a negative control, omit the DNA from the reaction. Use the negative control to determine the protein expression background in downstream analysis.

11. Use the S30 T7 Control DNA to synthesize Renilla luciferase, which has been codon optimized for use with mammalian cells. The expressed hRluc protein (37kDa) can be detected by Coomassie® blue staining of polyacrylamide gels following electrophoresis (Figure 2, Panel A, lane 2). The calculated molecular weight (MW) of hRluc protein is 37kDa, but it migrates with an apparent MW of ~33kDa.

12. The reaction may be incubated at 24–37°C. The fastest rate occurs at 37°C for approximately 1 hour, although the reaction will continue for several hours at a slower rate. Lower temperatures produce a slower rate of translation but often extend the time to several hours. Enhanced expression at lower temperatures for longer times appears to be gene- or protein-specific and may be tried if the standard reaction at 37°C for 1 hour does not produce the desired results.

13. To achieve maximum protein expression, quickly bring the reactions to the target temperature (e.g., place the reactions in a heat block preset to target reaction temperature), and shake the reactions vigorously at 300rpm with a floor incubator shaker or in a thermomixer at 1,200 rpm. 

Purification of His- or HQ-Tagged Recombinant Protein Using Magnetic Ni-Particles

Materials

• magnetic stand (see Section 10.C for options)

• Magnetic Ni Beads

• solid NaCl or 5M NaCl solution

1. Add 50µl of Binding/Wash Buffer to a 50µl reaction containing the His-tagged protein. [For HQ-tagged protein, also add NaCl (500mM final concentration) to improve binding of HQ tag to magnetic Ni-Particles.]

2. Vortex the magnetic Ni-Particles to obtain a uniform suspension.

3. Add 30µl of magnetic Ni-Particles.

Note: You may need to titrate the amount of magnetic Ni-Particles used for proteins expressed at higher or lower levels.

4. Invert tube to mix (approximately 10 times), and incubate for 2 minutes at room temperature. Make sure the magnetic Ni-Particles are well mixed.

5. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

6. Remove the tube from the magnetic stand. Add 150µl of magnetic Binding/Wash Buffer with 500mM NaCl to the magnetic Ni-Particles, and pipet to mix. Make sure that particles are resuspended well.

7. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

8. Repeat the wash step twice for a total of three washes.

9. Remove the tube from the magnetic stand. Add 100µl of magnetic Elution Buffer, and pipet to mix. Note: HQ-tagged proteins may elute at a lower concentration of imidazole (50–100mM) compared to polyhistidine-tagged proteins. The magnetic Elution Buffer, which contains 500mM imidazole, can be diluted with magnetic Binding/Wash Buffer or water to decrease the imidazole concentration, if desired.

10. Incubate for 1–2 minutes at room temperature. Place tube in a magnetic stand to capture the magnetic Ni-Particles. Using a pipette, remove the supernatant containing the purified protein.

Analyze the samples by SDS-PAGE or by functional assay. Note: Steps 8–10 can be omitted, and the sample containing the magnetic Ni-Particles can be used directly for SDS-PAGE analysis. Add 30µl of 1X SDS gel-loading buffer to resuspend the magnetic Ni-Particles. Load the sample directly onto an SDS-polyacrylamide gel.