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Affinity Tags and GST-Tag Properties

Recombinant protein expression performs in Escherichia coli (E.coli) where fusion protein binding with affinity tag is the most famous technique for purification of protein. Affinity tags are manufactured polypeptide chain which generally designed either N- or C- terminal sequence, by placing the cDNA sequence that encoded with tag peptide into an equal ORF of protein. Affinity tags have been promoted the folding of protein, solubility of protein and production of output in coupling to simplify purification of the recombinant process. There are some small size tags alike poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II- tags etc have the potential of decrease the impact of recombinant protein properties, activities and other function generally remove the affinity tag is not mandatory. Even more NusA, MBP, and Trx, GST and MBP show the leading solvency improving properties and protein expression efficiency as a large size tag in affinity chromatography technique. There are also more utilization of large size tags such as cell metabolic process, over-expression process and expression efficiency [12-14]. The glutathione S-transferase (GST) tag a is major founded and well-built up affinity tag based on the strong affinity of GST for immobilized glutathione during affinity chromatography technique when execute purification of recombinant protein. Glutathione affinity resins are easy to get from many commercial sources for purification of GST-tag fusion protein through their appropriate binding site to ligand, glutathione which is well-established immobilized on beadbased maintained. Glutathione affinity purification as like simple bindwash- elution representation is showed in the flowchart (Figure 2) [15]. GST-tag is best appropriate for utilize in prokaryotic expression since GSTs are a family of multifunctional cytosolic proteins that are show in eukaryotic living beings. In Escherichia coli (E. coli) some large size tags in affinity chromatography technique such as MBP tag and GST tags were utilized to enhance the analyzable of purified recombinant protein. By using reduced glutathione protein are eluted which carried GST-tag and captured by immobilized glutathione under non denaturing condition [16].

Figure 2: A Diagrammatic demonstration of Glutathione Affinity chromatography. Cleared lysate which carried GST fusion proteins that attach to immobilized glutathione (GSH, Bind). Non-binding proteins are discharge (washed) from the resin (Wash) and by the addition of excess reduced glutathione bound GST fusion proteins are eluted (Elute).

High efficiency construction of the target gene and pGEX gene fusion construction

In general, development of genes is expressed that presently cloned from cDNA which carries information of genome into vector such as pGEX or pET (Figure 3-I). This strategy was utilized by some researcher who developed a cDNA expression library expressed in Escherichia coli (E. coli). A small amount of clone expressed protein from cDNA expression library. Most of the inquiry reported that a few of gene were able to be coding partial gene sequence and some gene was not able to code the correct reading frame. In yeast, along these lines a new human cDNA expression library empowering the choice of the ORF establishment on histidine prototrophy was created and here this library around half of the clones were within the correct ORF [17]. Especially in the mammalian cells, there are some faults to the conformation of expression libraries. To the beginning the approach of non-translocate regions at both ends of clone for attaching fusion tag become critical situation. Another fault, although the method is difficult genes of interest must be habitually angled out of a library for utilize in test. For getting the target gene, polymerase chain reaction (PCR) is the most broadly used methodology and is always the primary step in any exertion to precise recombinant protein (Figure 3-II) [18]. Now-a-days by utilizing some online instruments approval of the gene and all primers can be designed. The PCR product refinement are presently developing the procedure that can be completed a computerized research facility in the laboratory [19]. Be that as it may, difficulty as a like band looks like nonappearance in agarose gel electrophoresis, non-specific groups and so on happen after PCR and moderate the experimental handle. By altering PCR parameter like change the annealing temperature, revised the concentrating of kits those issues can be overcome. There are some methodologies known as de novo synthesis of DNA which utilizing for getting the target gene (Figure 3-III). The DNA synthesis process as like solid phase which including chemical strategies has been customarily utilized but in this process [20 ], there are some difficulties arising. In addition, this process of DNA synthesis can reduce cost and impressively more for high efficiency about DNA construction. For synthesizing a long DNA sequence with expanded accuracy has been developed by used new array-based technique which showed significantly lower cost. De novo synthesis has some advantage, one of them that researcher can freely design the target gene without confinements utilize of common formats [21]. The reliable gene expression, increased protein products, protein solubility etc. are ensured quality using codon optimize gene. The development of this technique again and again, the pertinence of de novo DNA synthesis to the high efficiency measure is expected to extend. From GE Healthcare vectors such as pGEX gene fusion vectors has properties like the availability of a tac promoter for chemically inducible for bacterial growth and high-level protein expression with IPTG [22 ]. Some interior gene monitoring over an expression of insert by binding to the tac promoter until induction with IPTG. The polypeptide of interest can be inserted soon after the GST gene using a multiple linker site as like the pGEX gene fusion vectors are designed. Between the GST sequence and linker site there are an enzymatically digested site which is known as protease a cleavage site. GST gene molecule can be removed after digestion of protease enzyme as like thrombin. The linker site is carried by stop codon in each reading frame where encoded the stop codon sequence. One of the significances of using the thrombin cleavage site that is ordinary cost effective and during incubation at 25-37°C is enough for cleave between GST-tag and target protein. Moreover, when selecting a vector and picked up the restriction site that will be taken cleavage site of N-terminus sequence of the protein [23 , 24].

Figure 3: For making target genes it needs three types of different technique. (I) After reverse transcription target genes can be acquired from a cDNA library (II) amplify of genes from a cDNA library by utilization of PCR technique (III) Put together of small size of oligos can be employ to produce customized genes by the application of array-based oligonucleotide manufacture.

High efficiency cloning the GST fusion protein

Now days there are different kinds of process to create particular constructs for gene cloning and protein expression led to in a wide extend of living organism. Some cloning methods such as restriction enzyme based, ligation independent based and recombination-based cloning has empowered high-throughput protein expression for structural and functional analysis. These methods are moreover playing an important role by modification of the target gene, mutagenesis process and exchange of domain. Here we explain foremost common cloning procedures those are right now template for studies of gene cloning and recombinant protein expression, together with a brief outline of procedures related to co-expression technique [25].

Restriction enzyme-based cloning

Based on Restriction enzyme and DNA ligase it was the first convention accessible methodology is DNA cloning and in this method the restriction enzyme commonly utilized which recognition sequence that happen habitually inside open reading frames. The method of restriction enzyme-based cloning was already considered to be unacceptable for high efficiency although this cloning method performed with DNA ligation has been utilized for last some years. The most technique Flexi Cloning systems which was created by Promega Madison in the USA, where the two important rare cutting SgfI and PmeI were utilized and this technique has received expanded consideration since last few years. The two important link-up rare cutting SgfI and PmeI has been recommended to permit the cloning of maximum genes of chosen demonstrate life forms. The target genes are amplified utilizing primers containing adapter sequence and after that digested by two enzymes; this technique is following to that utilize for a common restriction enzyme-based cloning method [26].

Ligation-independent cloning

Ligation-independent cloning (LIC) is such kind of excessive result of cloning procedure that does not utilize restriction enzyme, which is available developed this process common and spontaneous. This procedure depends on the efficiency of bacteria cells to close the gap in DNA and the annealing of complementary single strand DNA. Ligation-independent cloning (LIC) is created a long time ago empowers directional cloning of any insert as a like single stranded complementary end which recognized after the generation of DNA fragments. Ligation-independent cloning is forming reasonable and effectively versatile to high efficiency due to need of necessity for DNA ligase and restriction enzyme. In any case, some enzyme such as T5 exonuclease and T4 DNA polymerase demand for the Ligationindependent cloning process to produce in vector or target gene using the single-stranded complementary ends [27].

Recombination-based cloning

Based on recombinant cloning technology the foremost popular high throughput cloning technology has been established and development of expression libraries some researcher performed this innovation day by day. In the site-specific of gene sequence recombinant cloning is negotiated by the genetic recombination enzyme and avoid utilize of restriction enzyme and DNA ligase. This type of genome areas is mostly made by long gene sequence which is makes extremely uncommon around 100 base pairs. The recombination area prepares to be cloned by insert DNA without anxiety of obstructing the coding sequence. Recombinase-based frameworks with high proficiency rates led to strong properties to exclusively review each target sequence. The cloning procedure such as Echo cloning (Thermo Fisher Scientific), Gateway (Waltham, USA) and creator (CA, USA) are broadly utilized as a recombination-based cloning system. In this system without utilizing any restriction enzyme and ligases, a site-specific recombination enzyme is utilized to develop the specified recombinant vector. Excessive result of cloning innovation, the most prevalent recombinant-based cloning technology has been utilized as a Gateway method which performs high efficiencies to succeed for all clones, adding cost and time through the project [28-30f].

Expression of GST fusion protein

The selection strains utilized to precise GST-tag recombinant proteins expression which leads to production and some expression plasmid used by GST-tag showed in (Table 2). Sometimes a couple of E. coli strains and its subsidiaries are broadly utilized for recombinant protein expression technique. Another strategy refers that, E. coli strains influence the expression of proteins that are encoded by a gene containing uncommon codons and in that case expression of proteins is dangerous to bacterial strain E. coli. In the post transitional modification process some gene makes strides by expression that can alter protein. A large strength behind the improvement of the GST fusion system is that the GST protein amasses in the cytoplasm and for a large size affinity tag solubility of GST moiety would be reproduced [31]. Production of GST recombinant protein which expressed in E. coli and performs manufacture of protein around 40mg/liter, but during the different situation as like unstable fusion protein does not yield higher amount. When expression levels are some larger, bacterial culture grown in shaker flasks, protein will be achieved from 3-4 liters of culture media [32]. The methodology of GST fusion proteins followed at 37 °C or 25 °C, the induction circumstance for each construct should be transformed to develop the maximum amount of protein production. The cultural media can be monitored optical density (OD) result 1.0 or 1.5 using A600 and finally, utilized of SDS-PAGE technique showed the molecular mass of expressed protein. During the bacterial culture added IPTG which induced the bacterial cell growth a particular time, as cell lysis can occur and in the cytoplasm release protease enzyme which can breakdown the GST fusion proteins [32].

Table 2: Some expression plasmid used by GST-tag affinity chromatography.

Purification of GST-tag recombinant protein used by affinity chromatography

By using an immobilized glutathione Sepharose column, GST-tag recombinant proteins are purified from bacterial cell lysates which suspension by PBS-buffer. Procedure of affinity chromatography is modifying assembled of glutathione Sepharose column using a roller/ tube pump to control flow rates. Phenylmethylsulphonyl fluoride (PMSF) is known as protease inhibitors and zinc metal (reducing agents) should be added to the buffers when suspension of bacterial cell pellets to decrease proteolysis of the GST-tag recombinant proteins. Protease inhibitors must be removed from the glutathione buffer prior to the enzymatic eradication of the GST-tag protein so that this kind of protease inhibitors will inhibit the proteolysis process. After purification of GST-tag recombinant protein to check out the position and molecular weight/mass by the analysis of SDSPAGE [34 ]. Sometimes another column chromatography used with a recombinant protein to to play down potential cross defilement which is confirmation of molecular mass of recombinant protein. Normally In additional, batch purifications which is known as speedy and basic, but habitually the abdicate and virtue of the protein acquired but sometimes this procedure obtained lower amount of recombinant protein compare with others chromatographic technique. For achieved purity of GST-tag recombinant protein purification result it would be better all experimental procedure carried out in cold room [35].

Enzymatic action to cleaved GST tag

After decided the expression vector, recombinant protein with GST-tag can be take off by the activity of protease enzyme (thrombin). This procedure happens at room temperature (25°C) either in protein solution in a falcon tube or attached to the chromatography column resin during chromatography running. This protease enzyme cleavage GST-tag and some good qualifications are related for example as like buffer, temperature, enzyme-to-substrate proportion, pH, length of hatching and concentration of protein etc. When GST-tag recombinant protein digested during column chromatography technique that time yield of digested protein is less amount but when incubation at 25°C, digested in buffer solution between GST-tag recombinant protein and thrombin is more effective, produced comparatively high amount and convenient. By used re-chromatography technique as like glutathion Sepharose column, target recombinant protein and GST-tag are separated [36, 37].

Further purification of remove GST tag and pooled recombinant protein

Without GST-tag pure recombinant protein purification steps, Glutathione Sepharose column used where recombinant protein carried non-functioning protease enzyme. The purified recombinant protein carried a little volume of GST-tag or other compounds/ fragments which did not attached glutathione resin during affinity chromatography technique. By used re-chromatography procedure, sometimes used ion exchange chromatography or S-100 (Size exclusion column) chromatography technique [38]. The purified pooled fraction of recombinant protein is usually more than 95% pure and analyzed the purity of recombinant protein by SDS-PAGE experiment which is showed in (Table 3). This recombinant protein can be stored below freezing for different purpose. An important strength of this research includes our review article (n=800). We can collect data consistently from last 10 years period, which increase the generalizability of the result. Furthermore, this study provides valuable information on protein technology which can be used to create new research question or hypothesis about GST-tag recombinant protein purification procedure although still now some limitation with this experiment [39].

Application of GST-tag affinity chromatography in plants sciences

Plants have been utilized as an originator of common items for last decades. It has various mechanical applications of plants that proceed to grow with the advancement of modern bioprocess and plant atomic science advances. For the expression of human therapeutics and biopolymers, one kind of methodology has been developed of plant transformation which driven to theutilize different types of plants moderator. The preliminary effective transformation in plant science occurs many years ago and plants have been identified as a viable substitute for recombinant protein production by GST-tag affinity chromatography. Plants recombinant proteins stages were at first ruled by rural crops with built up generation foundation such as tobacco, corn, rice including vegetables [40 -44]. A recent article reported that in plant molecular technique proposal has a handsome assembled pathway in short time that can achieve commercially acceptable recombinant protein production by GST-tag affinity chromatography. By the utilization of affinity chromatography various types of recombinant protein in especial field are produced from tobacco [45], Nicotiana benthamiana, and N. tabacum because of good protein expression and manipulation. Plants recombinant protein purified by GST-tag affinity chromatography has been emerged and contributed for human growth hormone production from tobacco cells which nominated later as patent being issued. Recombinant DNA innovation has made it conceivable to cross interspecies boundaries and achieved various types of plant assortments with alluring characteristics [46]. It plays an important role that by the used of this technology genetic modifications of crop plants mainly concern progressing their resistance and challenge to changing natural circumstance. Now days in plant molecular science field used some model plants for the purification of recombinant protein by the utilization of GST-tag affinity chromatography so that scientist can acquire knowledge in the field of genomics, transcriptomics, and metabolomics. In addition, as these plants are vulnerable to hereditary change, completely optimized conventions for recovery and development for perform of much important experiment and identify new idea is created for them [47f]. In any case, they have a few highlights that prohibit them from their planning utilize in different types of plant molecular research field. Some plants for example, the small size of Nicotiana tabacum or Arabidopsis thaliana seeds is more suitable and effective than leaves to produce recombinant proteins by the utilization of GST-tag affinity chromatography. Additionally, the extraction and purification of therapeutic proteins by GST-tag affinity chromatography from tobacco which is known as nicotine (one kind of alkaloid) that is toxic and harmful to human beings. There are multiple types of crops such as soybean, rapeseed, common bean, maize, wheat, and rice has been successfully utilized by recombinant protein purification for investigation of plants genetic engineering fields [48].

Update (Modified) development of GST-tag affinity chromatography in plants sciences

The affinity tags such as GST-tag has been modified which explained so far were improved to be the supporting point for recombinant protein purification by affinity chromatography where the presence of a complementary affinity ligand, immobilized in a separation matrix are necessary for this experiment. However, GST-tag can be capable and auto-sufficient for enhanced fusion protein solubility, purification and further characterization. For most of the affinity chromatography technique GST-tag are harmless and do not obstruct with the structure and biological function of the purified recombinant protein. Some researcher reported that the elimination of GST-tag can be executed by enzymatic cleavage or others chemical treatments showed extensively reviewed of recombinant protein purification technique. Sometime in plant science has some modified of GST-tag used in multimodal affinity chromatography which affinity ligands usually combine a hydrophobic moiety with ionic and hydrophilic groups, thus a perform on multi step purification schemes [49]. Moreover, another affinity ligand, anion-exchange and hydrophobic ligands are extremely popular as binding partners after changes/modify affinity GST-tags are widely employed for the purification of recombinant protein (Example: Zea mays and Rosa bourboniana). In addition, by the utilization of GSTtag affinity chromatography technique modified to structural ligands as binding partners of affinity tags or metal chelators as affinity ligands for His-tagged proteins perform a development of transformation and regeneration procedures for various plants recombinant proteins. The most implement of producing recombinant proteins in different fruits and vegetables (e.g., strawberries, bananas, potatoes, tomatoes, lettuce, spinach, rice, safflowers, and barley) has been investigated [50].

Future perspectives

Effective recombinant protein expression and decontamination is as often as possible vital for both fundamental investigate ponders and different types of biological implement. A large scale of protein expression and purification occurs in a bacterial strain named E. coli was started transformation in different investigate areas. Different types of laboratory work that were typically execute manually one molecular experiment at a time over 20-25 days can now be handled for the several molecular experiment in 7-8 days. In any case, some limitation is presently continued and in future advance changes are realizable with significantly. For example, analysis of specific molecule as like gene/DNA which used as a genetic sample for PCR [51], DNA microarray and next generation sequencing technique can be used in future for molecular diagnostics for drug design in pharmaceutical science, transformation of pathology and radiology field. Used by Gene cloning strategy a particular gene will be isolated that combine with vector genome for producing recombinant DNA which apply in future different agriculture field, plant disease, bio fertilizer, pesticides and pathogen identification with eradication. Others viewpoint such as gene cloning technology linked with post transitional modification technique has been developed for better understanding of molecular engineering in animal or plant genetic science that will apply as a great achieve in future at cell biology or others research field. However, the recombinant protein purification methodology will be used as medicinal research field such as vaccine, drugs or producing antibodies also in various biochemical analyses [52].

Conclusion

In this study we have demonstrated several key factors of GST-tag recombinant protein production and purification which frequently regularly vital for both essential investigate considers and various types of commercial applications. We have evaluated for the firsttime summarized data of GST-tag affinity chromatography to describe recombinant protein purification technique. GST-tag recombinant protein was over-expressed in E. coli used by different expression vector and the recombinant protein was purified by affinity chromatography technique and then cleaved with protease enzyme known as thrombin to remove the GST-tag. The recombinant protein was further purified by Glutathione affinity chromatography or anion-exchange chromatography technique. This GST-tag promote the recombinant protein solubility and stability. This review paper suggests that the application of GST-tag recombinant protein technology used as many beneficial purposes as possible of protein chemistry, structure analysis in NMR, 3D structure measurement, detection of functional group etc. Moreover, this GST-tag produces high amounts of homogenous pure fusion protein in a convenient way.

Ethical Approvals

This study does not involve experiments on animals or human subjects.

Data Availability

All data generated and analyzed are included previous research article. The current review covers a literature across from 1988 to 2020.

Author Contributions

Asaduzzaman Md, developed the concept, wrote the manuscript, and prepared the resources. Nahar Lutfun, took part in the manuscript writing and was responsible for the review. Lijon Md Bakhtiar, Imran Shahin and Rhaman Mohammad Saidur, have read and agreed to publish of this manuscript.

Acknowledgments

I would like to appreciate the advice and intellectual suggestions of all co-authors for their support, valuable suggestion and cordial companionship in making such an effort to submit the review manuscript. I am also express with deepest sense and thankful to all for their compatible manners, good assistance and unconditional cooperation.

Funding

This research received no external funding.

Conflict of interest

The authors declare that they have no conflict of interest. All the authors read and approved the final ma

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