Reasons for cleavage of protein after His-tag purification
It is important to carry out the cleavage of the resultant protein after a process of His-tag purification as the product may be having a myriad of proteins including raw lysate that originates from the bacterial host. These extra proteins may alter the purity of the resultant protein and affect any experimental procedures carried on the protein thereafter. In addition, some proteins may not have been denatured and as such, cleavage provides a way of getting rid of such resistant proteins. Some proteins possess activity that may, in turn, interfere with the proper application of the purified protein of interest. Cleavage generates different active proteins that can be utilized for different purposes. Furthermore, it acts to remove inactive or toxic residues from the protein of interest creating a pure utilizable protein or enzyme residue. Consequently, the by-products of cleavage may be utilized for different purposes in various industries be it biopharmaceuticals or the sugar industry.
Different sizes of proteins after His-tag purification
The process of His-tag purification may yield different sizes of pure proteins depending on the concentration of the salt used during the purification processes. High salt concentrations may result in low size proteins while low salt concentration yields proteins with higher sizes than expected. The type of salt also determines the size of the resultant purified proteins. Salts with more divalent cations may result in low-sized proteins. Some purified proteins are held in the flow-through and wash resulting in low-sized purified proteins while an overused or old matrix may result in incomplete purification leading to proteins with a bigger size than expected.
Reasons for using cell line BL21 DE3 for expression of protein better than cell line bl21 codon plus
The cell line BL21 DE3 has been employed in many purification processes of various forms of proteins. BL21 DE3 cell line is preferred to the cell line BL21 codon plus since it is a very versatile cell line and acts as an all-purpose cell line. In addition, it can be used in processes that entail the purification of advanced-level proteins as it provides an uncomplicated method of initiation of induction. The inductive process when using the BL21 codon plus is tedious and as such, unfavorable during purification of proteins using the His-tag technique. BL21 DE3 cell lines are highly compatible with numerous vector systems. Vector systems such as the pET vector system can be used with ease with this cell line as compared to the BL21 codon plus cell line. Consequently, the BL21 DE3 cell line provides a custom expression of the T7 vectors as compared to the BL21 codon plus cell line that provides for custom expression of non-T7 vectors. Because of these differences, the former is preferred in situations where His-tagging purification is required as a wide variety of vector systems can be employed.2 There is tighter control of some types of proteins that may be toxic when utilizing the BL21 DE3 cell line. In addition, the cell line can be modified to increase resistance to some types of antibiotics such as chloramphenicol.
Expression protein at 20 cº better on 37 cº
During the purification process using the His-tag method, expression is usually done at different temperatures depending on various factors. It is usually preferred to carry out the expression at lower temperatures around 20 0C rather than at 37 0C to prevent hydrolysis of the antibiotic present. Hydrolysis mainly occurs in antibiotics of the penicillin group such as ampicillin resulting in loss of antimicrobial activity. The loss in activity in turn results in proliferation and unregulated growth of cells that are devoid of plasmids. These cells proliferate in a manner such that they eventually overgrow the culture. In addition, lower temperatures foster increased solubility during purification facilitating effortless elution of the products of interest. Low temperatures offer protection to the reagents used as it prevents denaturation of the biomolecules used in the whole process of His-tag purification.
Concentration IPTG 0.1Mm better than 1 Mm TPTG
In the expression of proteins, there may be circumstances when a specific concentration of a particular reagent is required. In His-tag purification, it is usually better to use IPTG at a concentration of 0.1Mm rather than a concentration of 1Mm to prevent the formation of toxic protein residues in the host cells. In addition, higher concentrations of IPTG may lead to unwanted proteolysis of the various residues, and as such it is essential to use IPTG at a concentration of 0.1Mm rather than at a concentration of 1Mm. Consequently, there is a risk of the formation of inclusion bodies when IPTG is used at high concentrations. The inclusion bodies may result in isolation and collection of impure proteins and as such, interfere with the validity of the whole purification process.
Disadvantages of His-tag purification Batch chromatography
Batch chromatography is one of the chromatographic techniques employed in the protein purification process. This technique has some limitations that have curtailed its use. The binding of the protein of interest to the resin used is sometimes incomplete due to inefficient resin utilization, as a small portion of the resin is involved in the actual binding of the purified protein. Consequently, the rate of protein binding is altered due to sporadic injection of the feed and the whole process. Washing of proteins off the residue presents a problem since there is underutilization of the adsorbent. Elution of the protein of interest is problematic in batch chromatography as the whole product is in most cases highly diluted. Furthermore, the elution process requires large quantities of water to be effective. Batch chromatography offers low efficiency in advanced separating procedures such as the three-phase component separation process. Due to low loading factors, batch chromatography has limited productivity as compared to other forms of His-tag chromatographic techniques.
Advantages of His-tag purification FPLC
His-tag purification FPLC is advantageous, as it provides for highly purified protein residues compared to other forms of purification methods. FPLC has columns that provide for high-resolution separation of protein constituents. FPLC is a versatile method that can incorporate various buffers facilitating easier elution of the separated components.
Posttranslational modifications
Posttranslational modifications entail a transformation of a section or part of a purified protein by the use of special chemical reagents. In His-tag purification methods, post-translation modification entails the removal of glycoprotein remnants from the N and C-terminals of the histidine moiety to achieve the desired protein entity. This entails hydrolysis of amide bonds that link the glycoprotein to the core protein structure at the C-terminal of the histidine moiety. The products of post-translational modifications are detected using various spectrophotometric techniques such as mass spectrophotometry.
References
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Sussman, J & I Silman, Structural proteomics and its impact on the life sciences. World Scientific, Singapore, 2008.
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Roe, S, Protein purification applications: A practical approach. Oxford University Press, Oxford, 2001.