IVT vectors and purification strategies

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Choosing a vector and purification method for in vitro protein expression

Strategies for optimizing expression and purification of functional proteins produced using the Thermo Scientific 1-Step High Yield IVT protein expression system.

Eric Hommema, M.S.; Penny Jensen, Ph.D.; Krishna Vattem, Ph.D.; Brian Webb, Ph.D.;

October 22, 2013


In vitro protein expression is a rapid technique to produce functional proteins. By contrast with in vivo protein expression methods, in vitro protein expression allows for expression of toxic proteins, incorporation of heavy isotopes for NMR or MS analysis, addition of non-natural amino acids, and elimination of growth and induction conditions for recombinant protein synthesis.

The end goal of any protein expression system is production of highly functional and pure protein. Because every protein is different, no single purification strategy or protocol is optimal or successful for all proteins. In fact, the success rate for purification of proteins in any particular system is low. A collaborative database of cloned and purified proteins maintained by the Protein Data Bank lists a number of genes that have been successfully cloned and purified (Table 1).

Table 1. Number of successfully cloned, expressed and purified genes. Source http://targetdb.sbkb.org/statistics/TargetStatistics.html (January 2012).
. Cloned Expressed Purified % Purified
Prokaryota 147151 96750 38013 39%
Eukaryota 46588 27330 8019 29%
Archaea 14196 8812 3903 44%

Nevertheless, it is possible to identify certain techniques that can be used together to successfully express and purify a protein of interest. In this article, we describe, evaluate and recommend several workflows and troubleshooting tips for successfully purifying proteins expressed in the Thermo Scientific 1-Step High Yield IVT system.

Many vectors, many options

The 1-Step IVT system has specific vector requirements for high-level expression, all of which are found in the Thermo Scientific pT7CFE Vectors. These vectors contain an EMCV UTR upstream of the gene of interest, one to three affinity fusion tags, and a poly A 3’ tail. Affinity tags must be chosen with regard to the protein purification method to be used (Table 2), and each affinity tag has its particular advantages. For example, compared to the GST tag (26kDa), the His tag and antigen-based tags (HA, c-Myc, and FLAG) are advantageous when it is important to minimize the overall size of the recombinant protein, perhaps to maintain function.

Table 2. Available varieties of the Thermo Scientific pT7CFE1 Vector for protein expression by in vitro translation (IVT).
Vector N-term Tag C-term Tag Cleavage Site
pT7CFE1-NHis 6xHis
pT7CFE1-CHis 6xHis
pT7CFE1-NHA HA
pT7CFE1-CHA HA
pT7CFE1-NMyc c-Myc
pT7CFE1-CMyc c-Myc
pT7CFE1-NFtag FLAG
pT7CFE1-CFtag FLAG
pT7CFE1-NHA-CHis HA 6xHis
pT7CFE1-CGST-HA-His GST
HA
6xHis
HRV3C (C-term)
pT7CFF1-CGFP-HA-His GST
HA
6xHis
HRV3C (C-term)
pT7CFE1-NHis-GST-CHA 9xHis
GST
HA HRV3c (N-term)
pT7CFE1-NHis-GST 9xHis
GST
HRV3c (N-term)
Notes:

  • HA = YPYDVPDYA
  • c-Myc = EQKLISEEDL
  • FLAG = DYKDDDDK

The primary goal of our study was to learn if vectors with certain combinations of tags (and purification methods) would provide better overall results in terms of expression level and ease and quality of purification. We discovered that vectors containing an N- or C-terminal GST tag often provided greatly increased protein expression compared to vectors having only small antigen affinity tags. In addition, we found that the multi-tag pT7CFE vectors containing both GST and His tags provided greater flexibility and success for purifications. These multi-tag vectors accommodate the use of two different purification techniques, which may be used as alternatives or in combination (Figure 1). Inclusion of the HRV3c protease cleavage site provides for on-column or in-solution removal of the fusion tag.

 Figure 1. Purification options flowchart.

Figure 1: Purification options flowchart. Using a multi-tag vector that contains N- or C-terminal GST:His:HRV3c sequences enables any one of four alternative paths of purification to be used. The glutathione affinity workflow (bold path) is recommended.

In the next section, we provide the purification protocols outlined in the Figure 1. Then, in the subsequent RESULTS and DISCUSSION section, we present the results of our experiments that led us to recommend the glutathione affinity workflow. We also include a troubleshooting guide and suggestions for optimizing results for individual proteins.


PROTOCOLS:

GST-based purification using glutathione agarose

Immobilized glutathione offers minimal steps, high yield, and high purity under native conditions. The protocol is flexible allowing for elution of the entire fusion protein using 10-50mM glutathione or elution of the protein of interest free of fusion tags by using GST-tagged HRV3c protease. Buffer volumes listed are for purifications of 100µL IVT reactions (Part No. 88891). Scale the buffer volumes appropriately for 2mL (Part No. 88892) reaction volumes.

Protocol for GST-tagged protein expression and purification

Materials Required:

  • IVT cloning vector equipped with a GST tag and HRV3C cleavage sequence: pT7CFE1-CGST-HA-His (Part No. 88868), pT7CFE1-NHis-GST (Part No. 88870) or pT7CFE1-NHis-GST-CHA (Part No. 88871).
  • 1-Step High Yield IVT Kit (Part No. 88891) for 100µL reaction sizes. Scale the procedure for 2mL (Part No. 88892) reaction volumes.
  • Glutathione Binding Buffer: 125mM Tris, pH 8.0, 150mM NaCl, 1% Triton™ X-100, 10% glycerol, 1mM DTT
  • Glutathione Agarose Resin: Pierce Glutathione Agarose (Part No. 16101)
  • Spin Column: Pierce Spin Columns – Screw Cap (Part No. 69705)
  • Glutathione Wash Buffer: 125mM Tris, pH8.0, 1MNaCl, 1% Triton™ X-100, 10% glycerol, 1mM DTT
  • HRV3C Cleavage Buffer: 50mM Tris, pH7.4, 150mM NaCl
  • HRV 3C Protease (2 units/μL) that is GST-tagged: Pierce Human Rhinovirus 3C Protease (Part No. 88946)
  • Glutathione Elution Buffer: 10mM glutathione in Glutathione Wash Buffer

Procedure:

  1. Clone the gene of interest into the properly equipped pT7CFE1 vector.
  2. Express proteins with the 1-Step High Yield IVT Kit (Part No. 88891), following the procedure for a 100µL reaction, as described in the product instructions.
  3. Centrifuge the completed reaction (sample) for 1 minute at 10,000xg. Dilute the clarified sample 5-fold (1:5) in Glutathione Binding Buffer and add it to 200µL of Glutathione Agarose Resin in a Spin Column.
  4. Mix the sample end-over-end for 2 to 3 hours at room temperature. (Optional: Incubate 2 hours to overnight at 4°C for temperature-sensitive proteins.)
  5. Collect the unbound material by centrifugation for 30 seconds at 700xg.
  6. Wash the resin 3 times with 500µL Glutathione Wash Buffer.
  7. Elution Option A (Recommended): HRV 3C on-column cleavage
    1. Wash resin with 500µL of HRV3C Cleavage Buffer.
    2. Add 100µL of HRV3C Cleavage Buffer and 3µL of HRV 3C Protease. Incubate with gentle shaking (500rpm) at 4°C for 16 hours. Cleavage efficiency is protein dependent. To improve cleavage efficiency, increase the amount of HRV 3C Protease and incubation time.
    3. Collect protein by centrifugation for 20 seconds at 500xg. For maximum recovery, add an additional 100µL of the HRV3C Cleavage Buffer to the resin and collect the flow through via centrifugation. Because Pierce HRV 3C Protease is GST-tagged, it remains bound to the resin and the eluted protein is essentially free of protease.
  8. Elution Option B: Glutathione elution
    1. Elute the sample with 100µL Glutathione Elution Buffer (10mM glutathione in Glutathione Wash buffer).
    2. Collect the eluted material by centrifugation for 30 seconds at 700xg.

Cobalt IMAC purification of His-tagged proteins

Expressed proteins containing the His6x or His9x affinity tag can be purified by immobilized metal affinity chromatography (IMAC). Either cobalt- or nickel-based IMAC resins can be used for this purpose, but we have generally obtained greater purity using cobalt resin (data not shown). This protocol is effective for any His-tagged construct (N- and C-terminal ) that includes the HRV3C cleavage sequence; however, for best results, we recommend choosing one of the GST:His multi-tag vectors.

Protocol for His-tagged protein expression and purification

Materials Required:

  • IVT cloning vector equipped with the His tag and HRV3C cleavage sequence: pT7CFE1-CGST-HA-His (Part No. 88868), pT7CFE1-NHis-GST (Part No. 88870) or pT7CFE1-NHis-GST-CHA (Part No. 88871).
  • 1-Step High Yield IVT Kit (Part No. 88891) for 100µL reaction sizes. Scale the procedure for 2mL (Part No. 88892) reaction volumes.
  • Cobalt Binding Buffer: 100mM Tris, pH 8.0, 500mM NaCl, 1% Triton X-100, 10% Glycerol, 1mM DTT, 8mM imidazole
  • Cobalt IMAC Resin: HisPur Cobalt Agarose (Part No. 89964)
  • Spin Column: Pierce Spin Columns – Screw Cap (Part No. 69705)
  • Cobalt Wash Buffer (100mM Tris, pH 8.0, 500mM NaCl, 1% Triton X-100, 10% Glycerol, 1mM DTT, 10mM imidazole)
  • HRV3C Cleavage Buffer: 50mM Tris, pH7.4, 150mM NaCl
  • HRV 3C Protease (2 units/μL) that is His-tagged: Pierce Human Rhinovirus 3C Protease (Part No. 88946)
  • Cobalt Elution Buffer: 100mM Tris, pH 8.0, 500mM NaCl, 300mM Imidazole

Procedure

  1. Clone gene of interest into any of the His6X, C-GST-His-HA or N-His-GST expression vectors. For optimal expression and purification, the N-His-GST expression vector is recommended.
  2. Express proteins as described according to 1-Step High Yield IVT reaction instructions provided in kit (Part No. 88891).
  3. After completion of protein expression, centrifuge the sample for 1 minute at 10,000xg. Dilute the clarified sample 1:5 (100µL into 400µL) in Cobalt Binding Buffer and added to 25–50µL Cobalt IMAC Resin in a Spin Column.
  4. Mix the sample end-over-end for 1 hour a 4°C.
  5. Collect the unbound material by centrifugation for 30 seconds at 700xg.
  6. Wash the resin 3 times with 400µL of Cobalt Wash Buffer. Increase the number of washes and imidazole concentration (see subsequent Figure 6) to increase purity.
  7. Elution Option A: HRV 3c on-column cleavage
    1. Wash resin with 500µL of HRV3C Cleavage Buffer.
    2. Add 100µL of HRV3C Cleavage Buffer and 3µL of HRV 3C Protease. Incubate with gentle shaking (500rpm) at 4°C for 16 hours. Cleavage efficiency is protein dependent. To improve cleavage efficiency, increase the amount of HRV 3C Protease and incubation time.
    3. Collect protein by centrifugation for 20 seconds at 500xg. For maximum recovery, add an additional 100µL of the HRV3C Cleavage Buffer to the resin and collect the flow through via centrifugation. Because Pierce HRV 3C Protease is GST-tagged, it remains bound to the resin and the eluted protein is essentially free of protease.
  8. Elution Option B: Imidazole elution.
    1. Elute 3 times with 1 resin-bed volume (25 to 50µL) of Cobalt Elution Buffer.
    2. Collect the unbound material by centrifugation for 30 seconds at 700xg.

RESULTS and DISCUSSION:

Vectors with N-terminal GST tag provide highest expression

To evaluate the effect of tag size and type on expression, we compared expression levels for several proteins cloned in vectors with either a solitary C-terminal GST tag (large) or a solitary C-terminal HA tag (small). Depending on the protein, one or the other tag supported higher expression (Figure 2a). However, when the yield was greater with the GST tag, it was much greater indeed. This suggests that researchers should test new proteins with and without the GST tag to ensure that they do not miss the opportunity to achieve this high-yield expression.

We compared the effect on expression of N-terminal vs. C-terminal GST tag. For nearly all proteins tested, the N-terminal tag supported higher protein expression than the C-terminal tag (Figure 2b).

Figure 2a. Tag construction affects protein expression levels.

Figure 2b. Tag construction affects protein expression levels.

Figure 2. Tag construction affects protein expression levels in the 1-Step Human High-Yield IVT system. A. Comparison between GST and HA pT7CFE vectors (both C-terminal). B. Comparison between N- and C-terminal GST pT7CFE vectors (both of which also contain C-terminal HA tags). Selected pT7CFE plasmids (3.6μg) containing various gene sequences were added to a 100µL High-Yield IVT reaction and incubated for 17 hours at 30°C. An aliquot (2µL) of each translation reaction was analyzed via SDS-PAGE followed by transfer to nitrocellulose. Western blots were performed using the Pierce Fast Western Dura Kit (Part No. 35070) and target-specific antibodies for Figure 2A and anti-HA tag antibody for Figure 2B. The blots were visualized by the myECL Imager (Part No. 62236) and densitometry was obtained of the luminescent signal at the proper molecular weight using myImageAnalysis software (Part No. 62237).

Glutathione-based affinity purification provides greatest yield and purity

For purification of IVT-expressed proteins, satisfactory results can be obtained using either glutathione (for GST tag) or cobalt (for His tag) affinity resins. Multi-tag vectors containing both GST and His tags (as well as an HRV 3C cleavage site) provide for use of multiple elution protocols. The standard small molecule elution methods (i.e., 10-50mM glutathione for immobilized glutathione elution, or 300mM imidazole for cobalt elution) can be used in combination with on-column or solution-based cleavage with the HRV 3C protease.

We obtained the highest yield and purity by glutathione affinity purification (Figure 3). We cloned eight different proteins into both N-terminal and C-terminal multi-tag pT7CFE expression vectors. Then we expressed and purified them using either glutathione agarose resin or cobalt agarose metal affinity chromatography (IMAC) resin. Proteins were eluted from the columns by either 10mM glutathione or 300mM imidazole, respectively. All eight proteins were successfully purified by glutathione agarose using the N-terminal GST tag, while only six of eight proteins were successfully purified by cobalt agarose using the His tag (i.e., p53 and cFOS were unsuccessful).

Figure 3. GST-based vs. His-based purification of expressed proteins

Figure 3. GST-based vs. His-based purification of proteins expressed using the High Yield IVT. Eight different proteins were expressed with either an N-terminal (N) or C-terminal (C) His:GST fusion tag and purified using glutathione agarose (G) or cobalt IMAC  agarose (H). Proteins were eluted in G and H methods with either 10mM reduced glutathione or 300mM imidazole, respectively.

We improved overall purity in glutathione purification by eluting the proteins with on-column digestion with HRV 3C protease containing a GST fusion tag (Part No. 88946). For example, as shown in Figure 3, there is a 50kDa endogenous glutathione binding protein (elongation factor 1-gamma, EF-1g) that co-elutes with the GST-tagged protein of interest [1]. However, elution from glutathione agarose resin by cleavage with HRV 3C protease prevents this type of co-elution because only the recombinant protein contains the HRV 3C cleavage site. We tested five different proteins purified by glutathione agarose to compare elution by HRV 3C Protease (plus) or glutathione (minus) (Figure 4). Because the HRV3C protease contains a GST fusion tag, only the protein of interest is eluted while the GST-HRV3C protease and the cleaved GST fusion tag remains bound to the column.

Figure 4. Glutathione vs. HRV 3C protease for elution of GST fusion proteins.

Figure 4. Glutathione vs. HRV 3C protease for elution of GST fusion proteins. N-terminal GST fusion proteins were purified using glutathione agarose. Elution was performed as described in the protocol with 10mM glutathione or HRV 3C protease. In the lanes for Bad protein, the additional bands (triangle) are 14-3-3 proteins, which co-elute with Bad. Protein identification was verified by mass spectrometry (data not shown).

Imidazole affects IMAC-based purification of His-tagged proteins

Based on experiments comparing Ni-NTA and cobalt resins (data not shown), we recommend cobalt- over nickel-based IMAC for purification of His-tagged proteins expressed using the 1-Step Human High Yield IVT system. Cobalt provides a much higher level of purity than nickel, while at the same time maintaining good yield.

Compared to bacteria (the usual system for protein expression), the human proteome is larger and more complex. Consequently, human cell lysates (as in the 1-Step Human IVT system) generally result in higher background (co-elution of more off-target proteins) in IMAC than bacterial lysates. Besides using cobalt resin instead of nickel resin, we optimized for purity in cobalt-IMAC by adjusting the number of wash steps and the concentration of imidazole in the wash buffer (Figure 5). Increasing the number of washes with higher concentrations of imidazole resulted in greater purity, albeit with decreased yield.  Researchers can adjust these wash conditions to obtain an acceptable balance of purity and yield.

Figure 5. Purity and yield are affected by imidazole wash conditions buffer.

Figure 5. Purity and yield are affected by imidazole wash conditions buffer. Plasmid DNA (3.6μg of pT7CFE1-GFP-CHis) was added to 100µL of a high-yield reaction mixture with expression at 30°C for 18 hours. The Reaction mixture was centrifuged for 5 min at 5000xg and the supernatant was diluted 1:5 in Binding Buffer (100mM Tris, pH8.0, 500mM NaCl). The sample was added to 25µL cobalt resin in a spin column (Part No. 69705) and mixed end-over-end at 4°C. After incubation, the column was centrifuged for 30 seconds at 700Xg to collect flow through material. The resin was washed with various numbers of washing steps (400µL) and variable imidazole concentrations (Binding Buffer plus either 8 or 16mM imidazole). One third of the elution fraction was loaded onto a 4-20% SDS-PAGE gel and stained with Imperial Protein Stain (Part No. 24615). Percent yield is the measured fluorescence (ex/em = 510nm/485nm) of the (eluted fraction / crude fraction ) X 100.

Based on overall protein purification success and purity, immobilized glutathione affinity followed by on-column GST-HRV3c elution is the recommended workflow (Figure 1).

Purification optimization and troubleshooting

Protein purification success is highly protein-dependent. As described above, satisfactory results for glutathione and IMAC systems can be achieved with simple binding and wash buffer formulations based on Tris-buffered saline (TBS). Certain additives have the potential to increase purity or yield (Table 3). Other protocol modifications may help to troubleshoot difficult-to-purify proteins (Table 4).

Table 3. Recommended purification buffer additives.
Additive Function(s) IMAC Glutathione Resin
NaCl/KCl Protein solubilization, decrease non-specific binding 150 to 600mM 150 to 600mM (binding)
150 to 1150mM
(washing)
Imidazole Decrease non-specific binding in IMAC purification 5 to 15mM (Co);
10 to 40mM (Ni)
N/A
Non-ionic detergents (Triton X-100, NP-40) Protein solubilization; Decrease non-specific binding; Protein disaggregation 0 to 1.2% 0 to 1.2%
Reducing agents (DTT) Prevent protein aggregation 1 to 5mM 1 to 10mM
Glycerol Protein solubilization; Protein stabilization; Decrease hydrophobic interactions 0 to 20% 0 to 20%
Ionic detergents (CHAPS) Protein solubilization; Protein disaggregation 0 to 1% 0 to 1%

 

Table 4. Troubleshooting.
Problem: Low protein expression
If caused by poor plasmid preparation quality:

  • Ensure plasmid preparation is free of nuclease. Plasmid DNA should be precipitated with ethanol prior to use in the 1-Step Human in vitro system

If caused by improperly stored or expired kit reagents:

  • Ensure that kit components were stored at the proper temperature.
  • Test expression level using the GFP positive control to check expression system activity.

If caused by vector cloning error:

  • Ensure expressed protein contains expected fusion tag in frame. Confirm with an anti-fusion tag antibody in a Western blot.
Problem: Low purification yield
If caused by protein failing to bind to purification resin:

  • Clone protein of interest into N or C-terminal GST fusion vector.
  • Tag or protein has unfolded or aggregated. Express protein at lower temperatures (20-26°C) and for shorter duration.
  • Include non-ionic detergents (Triton X-100 or NP-40) during binding, washing, and elution steps.
  • For GST purification, add 1-10mM DTT to binding, wash, and elution buffers.
  • Increase the contact time with purification resin. Increase to over-night binding at 4°C.

If caused by protein remaining bound to column:

  • Perform all purification steps and maintain purification buffers at 4°C.
  • Purification resin dried out during purification. If using a spin column protocol, use short (30 sec) spin cycles.
  • For glutathione purification, increase glutathione to 50mM and collect multiple elution fractions. For cobalt IMAC, increase imidazole concentration to 500mM and collect multiple elution fractions.
  • HRV3c elution: Increase HRV3c concentration and increase cleavage time.
  • HRV3c elution: Fusion tag was not cleaved due to steric hindrance of the HRV3c cleavage site and the protein of interest. Switch to vector with the fusion tag on the opposite terminus.

 


CITED REFERENCES:

  1. Stergachis, A.B., et al. (2011) Rapid empirical discovery of optimal peptide for targeted proteomics. Nature Methods. 8(12):1041-3. [Describes purification of over 800 GST-fusion proteins expressed in the 1-Step Human IVT system. Purification methods and identification of impurities found in the protein elution fractions by mass spectrometry.]

GENERAL REFERENCES:

  1. Mikami, S., et al. (2008) A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expression and Purification. 62:190-198. [Describes alternate purification techniques for purification of expressed proteins from a human based in vitro high-yield expression system.]