The Denaturing and Renaturing are Critical Steps in the Purification of Recombinant Protein in Prokaryotic System

Proteins were first described in 1838 by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jons Jacob Berzelius. 1 The term protein comes from the Greek word proteios, meaning "primary". 2 Protein carries primary functions of the organisms, including both structural and physiological functions. One of the appropriate samples of its importance is the enzyme, which catalyzes almost all biochemical process in vivo. 3 Actually, lots of the most vital biological functions of an organism are fulfilled by proteins, so the isolation and expression of proteins become an important issue for both research and application.


Introduction
Proteins were first described in 1838 by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius. 1 The term protein comes from the Greek word proteios, meaning "primary". 2 Protein carries primary functions of the organisms, including both structural and physiological functions. One of the appropriate samples of its importance is the enzyme, which catalyzes almost all biochemical process in vivo. 3 Actually, lots of the most vital biological functions of an organism are fulfilled by proteins, so the isolation and expression of proteins become an important issue for both research and application.
Most proteins consist of 20 different L-α-amino acids, which are linked by peptide bonds, and finally form linear chains. There are 4 levels of structures of a protein. The primary structure of protein lies in the sequence of amino acids, the secondary structure of protein means the basic elements like regularly repeating local structures stabilized by hydrogen bonds, the tertiary structure of protein is its 3-demintional structure, and the quaternary structure exists in some proteins, in which a few single polypeptide chains aggregate together and function as a whole complex. 4 The sequence of amino acids in a protein is defined by the sequence of its genetic code. 5 As a commonly accepted opinion, the primary structure of a protein determines its higher level structure; however, the biological function of a protein is not decided by the amino acids composition, but most by its 3-dimentional structure. That is also why, not like DNA or RNA, to know the exact sequence of amino acids of a protein cannot ensure the successful production of the biological active one. During expression, isolation or purification processes of a protein, the natural structure of the protein must be preserved or recovered for it to exhibit normal biological activities. In this meaning, to develop a general technology or a method that can be used to purify every protein becomes an impossible task.
The production of recombinant proteins in prokaryotic system is a powerful tool that has been developed in the research and production of functional proteins for many years. To www.intechopen.com achieve successful production of a recombinant protein, the genetic code which decides the final amino acids sequence in the target protein is firstly sequenced. A vector containing the coding sequence is then cloned or synthesized, and transduced into bacteria, such as Escherichia coli (E. coli). The protein synthesis system in bacteria then recognizes the specific sequence, and proteins will be produced by the bacteria. Since the post-translational system are different in bacteria than the protein's original organism, the over-expressed recombinant protein are normally not functional, and will aggregate to form inclusion bodies. The inclusion bodies are easily to be isolated, and functional proteins will then be recovered from these inclusion bodies by different denaturing and renaturing techniques. Fortunately, there are some basic principles that can be applied to the expression and purification of protein, and in this chapter, some of them will be discussed. 6 In general, to achieve successful expression and purification of recombinant protein, the usual steps will be (1) obtain target gene; (2) prepare the expressing vector; (3) clone the target gene into the expressing vector with accuracy ensured by sequencing; (4) transduce host bacteria; (5) induce the expression of target protein; (6) analyze the expressed proteins; and (7) cultivate the engineered strain in large scale and (8) purify the expressed protein.

The purpose of target protein determines its form
In order to purify a protein, one must first ask themselves a question: what is the purpose of the target protein? If some wants to investigate the biological functions of a protein, it's appropriate to express and purify the protein in its full length or matured form, with natural bioactivity. Or, if the target protein is for antibody production, segment of the protein will serve the purpose well. Since proteins are synthesized as linear polypeptides and undergo post-translational modification and maturation, the denaturing and renaturing must also be considered in the expression and purification of recombinant proteins.
In order to investigate the biological functions of a protein in vitro or in vivo, full length, matured proteins need to be expressed and purified. One good example that can be easily produced and purified in E. coli is recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) 7 . rhIL-1Ra can be produced by E. coli at large scale in soluble form. The recombinant protein in supernatant of the lyzed bacteria can be purified by 2-steps of ionexchange chromatography to yield high-output biological functional proteins. However, some other proteins, such as recombinant human Midkine (rhMK), 8 recombinant mouse monokine induced by IFN-γ (rmMig) 9 , recombinant human Reg 4 (rhReg-4) 10 or recombinant human Chemerin (rhChemerin), 11 easily form inclusion bodies in E. coli, hence, necessary denaturing and renaturing steps must be applied. In some other cases, like recombinant murine CXCL14 (rmCXCL14), 12 full length protein is hard to be purified, and an additional tag will help to get final products with high yield and high purity.

Information collection
Once the target protein is decided, the initial step of the expression and purification of a protein is to capture target gene of a protein. This can be simply achieved by acquiring corresponding information of the genetic code of the gene that determines the amino acids sequence of the target protein from some database. The fragment of target genes can be easily synthesized or cloned, and cloned into different commercial available expressing vectors. The packed vector, containing the target gene, the selective markers and the elements needed for expressing in bacteria is then cloned into expressing strains, such as E. coli BL21 (DE3). Once the engineered strain is successfully constructed, it will then use its own resources to produce the alien protein.

The vectors and strains are the keys ensuring successful expression and purification
E. coli was discovered by Theodor Escherich in 1885. 13 It is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of endotherms. 14 E. coli is one of the most adopted bacteria in the expression and purification of recombinant protein, since it can be easily cultivated in laboratory in a relative cheap way, and has been intensively investigated as a widely studied prokaryotic model organism.
The pET system is developed by Novagen Company as a powerful system for the cloning and expression of recombinant proteins in E. coli. In this system, target genes are cloned in pET plasmids under control of T7 transcription. The expression is induced by T7 RNA polymerase in the host cell. When fully induced, target protein may consist more than 50% of the total cell protein in a few hours after induction. On the other hand, in the un-induced state, the expression level of target gene is low. pET system and its host bacteria E. coli BL21 (DE3) can be used as an optimized system in the expression and purification of recombinant protein. In order to choose a suitable pET system, a good source of knowledge comes from the manual of pET system offered by Novagen. 15

The cultivation conditions of E. coli should be tested and verified
Although the expression of recombinant protein in E. coli is normally high and consists large part of total bacteria protein, the expression could also be low, and the cultivation conditions of bacteria must be tested, including different temperature, vortex speed and induction conditions. Soluble, highly expressed target proteins are priorities in some cases, because the yield of target protein is high, and the biological activity is normally preserved; however, the downstream purification may be difficult, because the target protein is mixed with all soluble bacteria protein. Another side effect is that target protein may be digested by protease. On the other hand, if the target protein exists in inclusion bodies, it is non-biological active, but can be easily isolated with high purity, and the recombinant protein will not harmed by the presence of protease. After the optimized denaturing and renaturing processes, the downstream process may be much easier than soluble expressed target protein. In each case, the expressing condition must serve the downstream purification steps.
Normally, the conditions that lead to high growth speed and high expression result in aggregation of target proteins, for there are short time for the protein to fold correctly and so www.intechopen.com the inclusion bodies will be formed. Strong induction condition easily leads to highexpressing of target protein, and also leads to the formation of inclusion bodies. 16 On the contrary, lowering the cultivation temperature, adding some additives in cultivation medium and change medium can reduce the formation of inclusion bodies, and the target protein will present in soluble form. 17

The extraction of crude protein from cell lysates
Different methods can be applied to isolate the target proteins from the cell lysates maximally. Normally the first step after the induction of target protein is to lyze the bacteria. In this step, the target protein should be verified by Western Blotting using certain antibodies, and the proportion of the target protein in total cell lysates should be calculated. If the expression level of the recombinant protein is too low, the downstream process will become very difficult. Optimization needs to be considered to enhance the production of the target protein.
The goal of the extraction of recombinant protein is to facilitate the downstream process. The more concentrated proteins in this step, the easier the later purification will be.

The purification of soluble recombinant protein
If the protein exists in soluble form, it will probably possess natural bioactivity. The protein then needs no denaturing and renaturung treatments. For instance, when packed into pET11a expressing vector, rhIL-1ra is induced in a soluble form in E. coli. A good example of how to express and purify soluble rhIL-1Ra is indicated below. 18 The interleukin-1 receptor antagonist (IL-1Ra) is a protein encoded by the IL1RN gene. IL-1Ra was initially called the IL-1 inhibitor and was discovered in 1984. 24 IL-1Ra binds to the cell surface interleukin-1 receptor (IL-1R) hence blocks IL-1 signal pathway.
The full length of secreted rhIL-1Ra gene (gi: 186385) is 534bp with a CDS of 534bp, whereas the first 75 bps encode a 25-amino acid signal peptide. A DNA fragment corresponding to bp 76 to 534 in the gene IL1RN which encodes 152 amino acid residues in the matured rhIL-1Ra is cloned and packed into pET11a for the protein expression in E. coli. After induction, rhIL-1Ra mainly exists in the supernatant of the cell lysates.
The detailed processes of the expression and purification of rhIL-1a are listed below: The supernatant after ultrasonication was collected. 0.284 g ammonium sulfate was used for each milliliter of supernatant to precipitate rhIL-1Ra. Precipitated protein was solubilized 10-fold into the loading buffer 1 (1 mM EDTA, 20 mM Tris-HCl, pH 9.0). After centrifugation the supernatant was collected and named as rhIL-1RaST (Fig. 1

The denaturing and renaturing of recombinant protein in inclusion bodies
If the protein exists in inclusion bodies form, denaturing and renaturung treatments are mandatory for the purification of this protein.
An ideal denaturing and renaturing system must serve the following purpose: high-output, low-loss and normal bio-activity final products. In industrial level, the ideal renaturing technology must also be easily amplified to produce large-scale products. Must commonly used denaturing solution is 6 M guanidine chloride or 8 M urea. There are some renaturing methods including dialysis, dilution, ultra-filtration. Some additives can be applied into the renaturing system to enhance the refolding efficacy such as denaturing agents, PEG, detergents, redox system, L-Arg, chaperone, etc. However, there are no general guidelines that can be applied to all refolding systems and one must test the efficiency of each method to ensure the optimized output. Some commercial available kits can be used to test the optimized renaturing system.
Here are a few samples of protein expressed in inclusion bodies: Monokine induced by IFN-γ (Mig) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. Mature murine MIG is a protein of 106 amino acids with molecular weight of 14.4 kDa. It contains 4 cysteines and 2 disulfide-bonds. The mature mouse MIG cDNA covers the coding sequences of the gene without the signal peptide coding sequences. After induction, rmMig mainly exists in inclusion bodies of the cell lysates. 19 www.intechopen.com Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23. It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. Mature human Chemerin is a protein of 137 amino acids with molecular weight of 16 kDa. The mature human Chemerin cDNA covers the coding sequences of the gene without the signal peptide coding sequences and the last 21 bps coding the peptide need to be removed during maturation. After induction, rhChemerin mainly exists in inclusion bodies of the cell lysates. 20 Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily and was firstly isolated Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor. The mature human midkine protein is a protein contains 122 amino acids with a molecular weight of 17 kDa. The mature form of human midkine is encoded by the full-length sequence from 289 to 654 bp without the signal peptide coding sequences. After induction, rhMidkine mainly exists in inclusion bodies of the cell lysates. 22 Mouse CXCL14/BRAK is a monocyte-selective chemokine which is expressed in almost all normal tissues. The mouse cxcl14 mRNA encodes 99 amino acids, in which N-terminal 22 amino acids serve as a signal peptide which is cleaved as the protein is secreted. The remaining C-terminal 77 amino acids construct the mature chemokine with a calculated molecular weight of 9.4 kDa and pI 9.9. The mature form of mouse CXCL14 is encoded by the full-length sequence from 445 to 678 bp without the signal peptide coding sequences. After induction, rmCXCL14 mainly exists in inclusion bodies of the cell lysates. 23 The detailed processes of the denaturing and renaturing of those proteins were already published, and can be brifly introduced below Table 1.:

The purification of recombinant protein: Chromatography technologies
Like the denaturing and renaturing, lots of technologies had been developed to purify recombinant. Among which chromatography technologies are the most powerful tools, and can give satisfactory purity and yield of final protein products. According to the proteins pI, Ion exchange chromatography can be adopted. According to the hydrophilic and hydrophobic properties and molecular size, HPLC can be used. pET system carries certain fusion tag and can be easily identified by certain types of Affinity Chromatography. The most important guideline is, the purification of recombinant protein may not be achieved by simply using one technology mentioned above; on the contrary, the combination of them can lead to optimized results.
Like the above section, the detailed processes of the purification of the above-mentioned proteins were already published, and can be briefly introduced in www.intechopen.com 6. Purified recombinant proteins: The quality control and the storage 6

.1 The storage of recombinant proteins
Protein samples are easily to lose their bioactivity, so it's important to test appropriate storage conditions and buffers of this protein. After purification, the composition of storage solution and storage condition can both affect the bio-activity of recombinant proteins. For instance, rhIL-1Ra can be safely stored at 4°C for nearly half a year in 10 mM sodium citrate, 140 mM sodium chloride, 0.5 mM EDTA, while rhChemerin, when stored in PBS, pH 7.4, must be frozen in -80°C refrigerator. A good source of the knowledge of the suitable storage of the recombinant protein comes from the manual of commercialized recombinant protein.

The quality control of recombinant proteins
After protein expression and purification, some quality control items should be tested to serve the later in vivo and in vitro experiment, or other application. These test items include but not limit to: