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Research Article
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A Novel Thermostability Conferring Property of Cherry Tag and its Application in Purification of Fusion Proteins
Krishna Mohan Padmanabha Das, Shruti Barve, Sampali Banerjee,
Suman Bandyopadhyay and Sriram Padmanabhan*
Lupin Limited, Biotech R & D, Gat #1156 Ghotawade Village, Mulshi Taluka, Pune-411042, India
*Corresponding author:
Dr. Sriram Padmanabhan,
Director, Biotechnology R & D Lupin Limited,
Gat #1156, Ghotawade Village, Mulshi Taluka,
Pune-411042, India,
Tel: + 91-20-66549801,
Fax: + 91-20- 66549807,
E-mail: srirampadmanabhan@lupinpharma.com
Received December 08, 2009; Accepted December 26, 2009; Published December 26, 2009
Citation: Das KMP, Barve S, Banerjee S, Bandyopadhyay S, Padmanabhan S (2009) A Novel Thermostability Conferring Property of Cherry Tag and its Application in Purification of Fusion Proteins. J Microbial Biochem Technol 1: 059-063. doi:10.4172/1948-5948.1000012
 
Copyright: © 2009 Das KMP, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
Cherry tag, a red polypeptide of the heme binding part of cytochrome is used to attain high levels of soluble protein expression in E. coli. A novel heat stability conferring property of this tag was observed and studied for constructs of two soluble fusions especially Cherry-Granulocyte colony stimulating factor (GCSF) and Cherry- Staphylokinase (SAK). Heat incubation of these fusion proteins at 70°C for 20 minutes culminated in specific denaturation and precipitation of E. coli proteins excluding the fusion proteins. Both the heat treated fusion proteins were found to be functionally active. Thus Cherry fusion tag could be used as a cost-efficient tool in purification of proteins by imparting heat stability.

Keywords
Cherry vector; Fusion tag; Thermostability; GCSF; Staphylokinase; Heat treatment

Introduction
Gene fusion is a successful approach used for producing soluble heterologous proteins in E. coli (Uhl’en and Moks, 1990). A couple of carrier proteins that are widely used in gene fusions include thioredoxin (Trx), maltose-binding protein (MBP), glutathione S-transferase (GST), intein, calmodulin-binding protein (CBP), N utilization substance A (NusA), cellulose-associated protein (CAP) etc. In spite of the fact that the use of these carrier proteins has resulted in successful over expression of many heterologous proteins, each of these tags were assessed empirically and certainly do not possess maximum solubilizing features (Shih et al., 2002).

The Cherry™ Express vector, from Eurogentec, USA is one such vector which is being used for similar purposes. This vector possesses a small sequence encoding a red polypeptide of the heme binding part of cytochrome under T7 promoter coding for a protein of molecular mass 11 kDa. This tag favours the solubility of any protein fused to its C terminal portion and appears red on expression allowing one to explicitly and accurately quantify the concentration of target protein by a simple absorbance measurement at 413 nm. The red color constitutes a visual marker throughout protein purification and this tag is cleaved using enterokinase enzyme resulting in authentic N terminal sequence of the target protein (http://www.eurogentec.com/product/research-cherryexpress.html).

In an effort to attain large quantities of proteins for structural, functional and other characterization studies, we constructed pSCherry1-GCSF and pSCherry1-SAK cassettes and expressed them in a suitable bacterial host. We noticed a unique heat stable property of such fusion proteins. Since both native GCSF and SAK proteins are thermolabile, we investigated the advantages of use of the heat stability conferring property of Cherry tag on these proteins and implications of our findings are discussed in this paper.

High-temperature precipitation is a well demonstrated technique that is usually employed in the initial stages of purification of thermostable proteins (Takesewa et al., 1990; Kirk and Cowan, 1995; Ng et al., 2006). A distinctive use of protein heating was recently illustrated as a protein-purification polishing step to increase the diffraction quality of protein crystals (Pusey et al., 2005). The method is based on the assumption that improperly folded proteins are less heat-stable and have a higher propensity for aggregation than correctly folded proteins. Heating will thus precipitate misfolded proteins, thereby increasing the protein-solution uniformity of the residual protein and apparently the crystal quality (Karlsson and Eriksson, 2007).

To investigate the effect of heat treatment on the activity of the fused proteins of interest, we undertook a detailed study of purification and assessment of biological activity of one of the fusion proteins, Cherry-SAK, following thermal treatment at 70°C for 20 minutes and compared its purification profile with that of heat untreated Cherry-SAK fusion protein together with the untagged SAK protein.

Materials and Methods

Bacterial strains and plasmids
Escherichia coli DH5a cells were used for propagation of plasmids. The restriction enzymes were procured from Bangalore Genei Pvt. Ltd, Bangalore, India. BL21(DE3) and BL21(DE3) codon plus cells were purchased from Stratagene, USA while the oligos, PCR extraction kit and Genelute kit and other fine chemicals were purchased from Sigma, USA. The anion exchanger matrix Q Sepharose FF was from GE Healthcare, Sweden.

Cloning of pSCherry1-GCSF and pSCherry1-SAK constructs
hGCSF gene was amplified by polymerase chain reaction (PCR) using synthetic hGCSF gene as a template (Somani et al., 2009). The sequence of the forward and the reverse primers were 5´ CCG CCG GGA TCC GAT GAT GAT GAT AAA ACG CCA TTA GGC CCG GCC 3´ and 5´ CCG CCG GAA TTC AAG CTT TTA CGG CTC CGC TAA ATG ACG 3´, respectively. The digested PCR product was cloned into pSCherry1 vector as a BamHI/EcoRI fragment.

Staphylokinase gene was amplified by PCR from the synthetic SAK gene as described (Mandi et al, 2009). The oligos of the sequence 5´ CCG CCG GAA TTC CAT ATG TCA AGT TCA TTC GAC AAA GGA 3’ and 5´ CCG CCG GAA TTC AAG CTT TTA TTT CTT TTC TAT AAC AAC 3´ were used as forward and reverse primers, respectively. The amplified PCR product was purified and cloned into pSCherry1 vector as an NdeI/ HindIII fragment.

Shake flask expression studies of Cherry-GCSF and Cherry- SAK and sub cellular localization
pSCherry1-SAK and pSCherry1-GCSF clones were introduced into BL21 (DE3) Codon plus and BL21(DE3) cells, respectively and the protein expression was induced with 1 mM IPTG for 4 hours at 37°C. The induced cell pellets were resuspended in 10 mM Tris pH 8.0 at a 1:40 (w/v) ratio and subjected to lysis using a homogenizer (Nano De Bee, Bee Intl, USA) at 25,000 psi for 2 cycles followed by centrifugation at 13,000 rpm for 10 minutes to separate the soluble and insoluble fractions of the harvested cells. All the samples were later analyzed on SDS-PAGE followed by coomassie staining.

Heat treatment methodology and analytical methods
The clarified cell lysate supernatant was subjected to heat at 70ºC for 20 min containing 100 mM sodium chloride followed by centrifugation at 13,000 rpm for 10 min to separate the heat denatured and the heat stable protein fractions. These fractions were subsequently analyzed on SDS-PAGE (Kirk and Cowan, 1995).

The densitometry analysis of the protein of interest was carried out following the method of Catzel et al. (2003). Purified GCSF and SAK protein obtained from the pET21a constructs (Somani et al., 2009; Mandi et al., 2009) (data not shown) were also subjected to heat as specified earlier.

Purification of Cherry-SAK fusion and untagged SAK proteins
The soluble Cherry-SAK protein, with and without heat treatment, were dialyzed against 100 volumes of 10mM Tris pH 8.0 in cold for 16 h. The dialyzed samples were then loaded onto a Q-Sepharose (anion exchanger) column equilibrated at pH 8.0. After washing the column with 10mM Tris pH 8.0, the bound protein was eluted using a gradient of NaCl solution (0.1 to 1.0 M). Fractions comprising Cherry-SAK were pooled and analyzed on SDS-PAGE. Similar protocol was pursued for untagged SAK protein (without heat treatment) expressed from pET21a- SAK construct (Mandi et al., 2009).

Chromogenic assay of SAK
SAK activity was quantified using plasminogen coupled chromogenic assay as described by (Apte-Deshpnade et al., 2009). Briefly, 25 mU of human plasminogen, the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) and samples containing SAK were incubated in 100 μl reaction volume in 96-well flat-bottom plates (Nunc) at 25°C for 20 min. Amount of p-nitroaniline (pNA) released was monitored at 405 nm by plate reader (Multiskan Spectrum, Thermo, USA). One unit (1 U) of SAK is the amount of the enzyme needed to form 1 U of plasmin from plasmingen. Units of plasmin formed were estimated from the amount of chromogen (pNA) formed using a standard curve of pure pNA.

Biological activity of Cherry-GCSF using NFS-60 cells
In-vitro cell proliferation assays were performed to determine the biological activity of heat treated crude Cherry-GCSF fusion proteins as specified earlier (Somani et al., 2009) with the following modifications. Briefly, the NFS-60 cells were maintained in RPMI medium with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (1mg/ml), L-glutamine (2mM), sodium pyruvate (1 mM) and mouse IL-3 (33U/ml). For proliferation assay, cells were pre incubated for 3 hours in the above medium without IL-3. Subsequently, these cells (2.5 x 104cells/ 100μl) were treated with Cherry- GCSF fusion proteins (250 pg/ ml of GCSF equivalent) in 96-well tissue culture plates for 48 h at 37°C and 5% CO2. The cell proliferation was measured by CCK-8 Kit (Dojindo, Maryland, USA) as per manufacturer’s protocol. Briefly, 10 μl of the assay reagent was added and amounts of formazan formed (an indicator of the number of live cells i.e. biological activity) was estimated by measuring OD at 490 nm after additional 3 hours of incubation.

Results
The Cherry-GCSF and the Cherry-SAK fusion proteins were found to be localized into the soluble fraction of the cell as represented in the Figure 1 (lanes 3 and 4 respectively). The untagged SAK protein was also a soluble protein (data not shown) as described earlier (Mandi et al., 2009).

fig
 Figure 1: Expression of Cherry-SAK and Cherry-GCSF. SDS-PAGE profile of insoluble and soluble fractions of Cherry-SAK and Cherry-hGCSF followed by coomassie blue staining. Lane 1: Protein Molecular weight Marker (14-97 kDa) Lane 2: Insoluble fraction of Cherry-hGCSF, Lane 3: Soluble fraction of Cherry-hGCSF, Lane 4: soluble fraction of Cherry-SAK, Lane 6: insoluble fraction of Cherry-SAK.

Upon heat treatment, almost 90% of the Cherry-GCSF fusion protein appeared in the heat treated supernatant (Figure 2a, lane 3) while almost 100% of the SAK protein was present in the heat treated supernatant (Figure 2b, lane 6) as judged by densitometry scanning. The untagged crude SAK protein was, however found to precipitate completely (Figure 2b, lane 4) under similar heating conditions.

fig
 Figure 2: Heat denaturation studies in the presence of 0.1 M NaCl. Crude soluble fractions of SAK, Cherry-SAK and Cherry-GCSF were heated to 70°C for 20 minutes followed by centrifugation at 13,000 rpm for 10 min. The cleared heated lysates and the pellets were analyzed by SDS-PAGE followed by commassie blue staining.
A) Lane 1: Molecular weight marker(14-97 kDa), Lane 2: Soluble fraction of Cherry- hGCSF, Lane 3: Heat treated cleared supernatant of Cherry-hGCSF, Lane 4 : Pellet after heat denaturation. B) Lane 1: Molecular weight marker(14-97 kDa), Lane 2: Soluble fraction of SAK, Lane 3 : Heat treated cleared supernatant of SAK, Lane 4: Pellet after heat denaturation, Lane 5: Soluble fraction of Cherry-SAK, Lane 6 : Heat treated cleared supernatant of Cherry-SAK.

To gain further insight into thermostable nature of these fusion proteins, we proceeded with purification of Cherry-SAK fusion protein with and without heat treatment. The results indicated (Figure 3) that while the heat treated Cherry-SAK fusion protein showed a purity of 85% (Figure 3b, lane 4), the untreated fusion protein showed merely 35% purity (Figure 3a, lane 3) when purified in the same manner. It is important to emphasize at this stage that irrespective of heat treatment, the binding property of the Cherry-SAK, remained unaltered as reflected from the fact that under both the conditions the proteins were optimally eluted with 300 mM sodium chloride (Figure 3a and 3b). Under similar purification conditions when untagged SAK was loaded on Q Sepharose, the eluted untagged SAK showed merely 13% purity (Figure 3c, lane 3). This result is encouraging since it opens up a new method for purification of all Cherry fused proteins by the methodology followed.

fig
 Figure 3: Purification of Cherry-SAK with or without heat treatment and untagged SAK on anion exchange column. A) Cherry-SAK purified on Q Sepharose. Lane 1: Molecular weight marker (14-97 kDa), Lane 2: Soluble fraction of Cherry-SAK cell lysate, Lane 3: 0.3 M NaCl eluate of Q-Sepharose. B) Heat treated Cherry-SAK lysate purified on Q Sepharose. Lane 1: Molecular weight marker (14-97 kDa), Lane 2: Soluble fraction of Cherry-SAK cell lysate, Lane 3: Heat treated cleared supernatant of Cherry-SAK, Lane 4: 0.3 M NaCl eluate of Q-Sepharose. C) Untagged SAK purified on Q Sepharose. Lane 1: Soluble fraction of SAK cell lysate, Lane 2: Molecular weight marker (14-97 kDa), Lane 3: 0.3 M NaCl eluate of Q-Sepharose.

It was also intriguing to see that the Cherry-GCSF fusion protein after heat treatment displayed nearly 50% biological activity as shown in Figure 4a in comparison to NIBSC standard while there was no activity for the purified heat treated native GCSF. This suggested that Cherry tag indeed offers heat stability to GCSF when it exists as a fusion protein. Also, the SAK activity of the purified Cherry-SAK fusion protein (Figure 4b, sample 3) was found to be similar to the activity seen with the untreated Cherry-SAK (Figure 4b sample 1). It was interesting to note a marginal increase in the SAK activity for the heat treated Cherry- SAK preparation and this might be due to the deactivation of certain E. coli proteases upon heat treatment (Figure 4b, sample 2). It was also observed that more than 80% of the activity of the purified SAK was lost when it was subjected to the heat treatment under similar experimental conditions (Figure 4b, sample 5).

fig
 Figure 4: Activity assay of Cherry-GCSF and Cherry-SAK. A) Biological activity of Cherry-GCSF. Sample 1: NIBSC GCSF standard, Sample 2: Purified GCSF expressed from pET21a-GCSF construct, Sample 3: Purified GCSF heat treated at 70°C for 20 min, Sample 4: Untreated crude Cherry-GCSF fusion protein, Sample 5: Heat treated crude Cherry-GCSF fusion protein. B) Chromogenic assay of Cherry-SAK. Sample 1: Untreated crude Cherry-SAK, Sample 2: Heat treated crude Cherry-SAK, Sample 3: Heat treated purified Cherry-SAK, Sample 4: Purified untagged SAK expressed from pET21a-SAK construct, Sample 5: Heat treated pure untagged SAK.

Discussion
Fusion tags that allow enhanced recovery using economical purification methods are easily scalable for industrial downstream processing. Some of the fusion tags that promote secretion of target proteins are also useful tags based on assay on enzymatic activity or antibody binding (Ford et al., 1991). Since many fusion tags do not interfere with the biological activity of the target protein and in some cases have actually been shown to stabilize it, they are preferred for hyper expression of proteins (Banerjee et al., 2009). Nevertheless, for the purification of authentic proteins, a site for specific enzyme cleavage is often included, allowing removal of the tag after recovery.

We noticed a novel characteristic of Cherry™ tag in conferring heat stability to otherwise heat labile proteins and since this is being stated for the first time, we studied this property in detail. A couple of proteins have been purified using such heat stable properties of the fusion tags include purification of immunoglobulin G from rabbit serum (Osmark et al., 2003), recombinant antibodies purification from Camelidae (VHHs) (Olichon et al., 2007), heat purification of both RE3 and VHs (Marco et al., 2004) and purification and characterization of membrane protein di-geranylgeranylglyceryl phosphate synthase from archaea etc. (Roy et al., 2007). The inherent thermal stability of thioredoxin and its susceptibility to quantitative release from the E. coli cytoplasm by osmotic shock can also be exploited as useful tools for thioredoxin fusion protein purification (McCoy, 2001).

The temperatures that have been tried for purification of proteins by heat treatment protocol range from 50 to 80°C. The mechanism that has been attributed to such an effect is that the heat treatment causes permanent unfolding of proteins together with formation of aggregates due to its exposed hydrophobic core with increased entropy. In particular, the fusion proteins are denatured but can correctly refold after withdrawal of heat treatment and this feature enables their differential purification as heating denatures and precipitates thermosensitive bacterial proteins irreversibly (Kwon et al., 2008).

A statistical trend seems to appear on the amino acids that contribute to thermostability of proteins especially when one compares the amino acid composition of a mesostable protein with a thermostable protein. The ratio of number of glutamic acid and lysine to glutamine and histidine has also been shown to be a responsible factor for heat stable property of any protein (Farias et al., 2004). The heat stability of the protein is expected to be higher when such a ratio is higher than 2.5. The Cherry tag amino acid sequence exhibited a ratio of 3.0 by this criterion and hence it is tempting to speculate that the number of glutamic acid and lysine could be one of the possible factors responsible for the heat conferring property of the cherry tag reported here. It is interesting to note that the fusion tags such as NusA, GST have different heat stable ratio and hence this criteria could be used for choosing fusion tags for cloning and subsequent purification.

The observation of retaining the biological activity of the Cherry fusion protein even after heat treatment demonstrates that the proteins are folded in the right confirmation and indicates that the Cherry tag imparts heat stable property to the proteins illustrated here, since both these proteins are known to be susceptible to heat in their native and untagged state.

Heat purification, thus, is not only a fast and economical alternative to conventional chromatography but, when applicable, can also simplify the downstream operations in large scale manufacturing of the recombinant therapeutics. The efficiency of heat purification to eliminate the endotoxins and host cell protein (HCP) load from bacterial recombinant protein preparations also appears to be of prime significance.

In summary, we have developed a cost-effective method for purification of proteins expressed in E. coli using the heat conferring property of the Cherry tag. This vector enables us to screen highly soluble proteins effectively and also shows promising potential in purification of the proteins with a simple heat treatment approach, thus reducing the protein load to column chromatographic separations. Similar approach can be applied to alternative cloning of all potential target genes into vectors of different expression systems, including yeast, insect, and mammalian cells, as well as cell-free in vitro systems with this tag with suitable promoter systems. This method also appears well suited for automation and might be a useful tool for the production of proteins for structural and functional genomic studies.

Acknowledgements
The authors are thankful to Mr. Sachin Rewanwar, Dr. Bhaskarjyoti Prasad and Mr. Srinivasa Kumar for helping us with some experiments. Authors are also thankful to Dr. Kamal Sharma, Managing Director, Lupin Limited, India for his continuous encouragement and support.

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