Numerous proteins have previously reported to fold on their own in vitro and work done by [1], shows that linear polypeptides chain poses the required information which determines the three-dimensional structure of the protein. All the processes involved in protein folding have drawn a lot of attention for many years in the area of molecular biology. Amongst the other reasons is that most proteins lack the capability to fold on their own, thus aggregates and form inclusion bodies. Heterologous protein expression using the Gram-negative bacterium, E. coli, is an important and frequently used for genetic manipulations and for expressing recombinant proteins. Bacterial expression systems are widely used and favored by industrial and pharmaceutical companies because of their ability to rapidly produce recombinant proteins using low cost substrates [2]. Nonetheless, the overexpression of some recombinant proteins in E. coli may result in the formation of insoluble proteins [3] Most proteins including those from Plasmodium are expressed as insoluble proteins in inclusion bodies [4] Inclusion bodies primarily contain over-expressed nonnative proteins and occurs as a result of differences between the codon usage of the E. coli and the protein of interest [5]. Mehlin [4] selected and attempted to express 1000 P. falciparum open reading frames using E. coli BL21 Star™ (DE3) cells. Only 6.3% of these cells were expressed as soluble proteins. The same group attempted to improve expression by a technique called “codon-optimisation” an approach where codons of the target gene were altered to suit the codon preference of expression host [2] Codon-optimisation had no significant influence on the production of P. falciparum proteins from E. coli [4] Consequently, less than 0.5% of all protein structures available on the Protein Data Bank were malarial proteins [6,7]. Furthermore, the production of misfolded or partially misfolded proteins should also be avoided as they often exhibit a lower activity as compared to monomeric folded proteins and consequently the expression of misfolded protein adds to the production costs since they will require more purification steps and refolding procedures.

Conventional ways used to overcome inclusion bodies

Various buffer formulations using surfactants and amino acids (such as L-arginine) have previously been reported to suppress protein unfolding or aggregation [2]. However, the major restriction with the use of surfactants is that they tend to co-purify with the protein of interest. Although some studies have shown that the use of L-arginine suppresses protein aggregation [8], other researchers still argue that L-arginine may play a significant role on the protein thermodynamic stability [5,9]. The use of low incubation temperatures ( ≤ 20°C) have also been identified as important factors in the expression of soluble proteins [10] Various studies have demonstrated that the proteins expressed at low temperatures are likely to be more soluble [11,12]. Wang and his colleagues [12] hypothesized that at low temperatures the expression rate of proteins is greatly reduced, thus increasing the folding efficiency of protein. It was proposed that endogenous proteases may be less-active at low temperatures, thus, allowing the expressed protein properly fold [2].

Codon bias refers to the high frequency and preferential use of a particular codon coding for an amino acid within an organism [13]. There is a high level of codon mismatch between the protein of interest and expression hosts, such as E. coli and two different strategies have been used to minimize codon bias. Firstly, the intracellular tRNA pool can be expanded by using plasmids which encode rare tRNAs used in certain organisms, for example E. coli. Secondly, altering the codons of the target genes to suit the codon preference of the non-natural expression host is a process referred to as ‘codon-optimization’. This approach has met with some success, especially in antimalarial vaccine targets, but unfortunately the expression of several other malaria proteins was not improved through codon-optimization alone (Figure 1). As an example, Mehlin [4] reported that nine out of the twelve P. falciparum genes that had been synthesized with optimized codons could still not be expressed in E. coli, and the three proteins that were expressed were in inclusion bodies. This led them to conclude that codon usage did not have a significant impact on protein expression.

Figure 1:Commonly used strategies for recombinant protein production. Extensive sequence analysis of a gene could facilitate genetic manipulation of the gene if needed. E. coli should remain the starting point for the expression of soluble proteins due to its ease of use. Several optimizations including codon adaptation, chaperone co-expression and small sequence changes might be particularly necessary for the expression of the protein of interest. Various other expression hosts including mammalian, Yeast, S. cerevisiae, Pichia pastoris have been used with various levels of success and might include an iterative process incorporating necessary optimizations. In most cases, the final product is expected to be pure, soluble and active.

Engineered cells to improve protein production

Biologically active impurities can jeopardize research or therapeutic applications even if present in trace amounts. One consequence during purification of proteins expressed in E. coli is DnaK contamination [6]. One approach developed to circumvent DnaK contamination is extensive washing of columns with ATP since DnaK in its ATP-bound state has low affinity for protein. However, this strategy lengthens the purification procedure and is immensely expensive for large scale purifications [14]. In an attempt to eliminate DnaK contamination it was investigated whether recombinant proteins could be produced in the absence of DnaK. Towards that end, the ΔdnaK derivative of the extensively employed E. coli B host strain BL21 (DE3) was constructed. The consequences of the absence of DnaK for the production, solubilize, correctly assembly, and activities of several recombinant proteins in BL21 (DE3) have been studied. Obtaining a BL21 (DE3) ΔdnaK strain has allowed to elucidate to what extent such an E. coli chaperone is indispensable to protein overproduction in the particular genetic context of an E. coli strain that lacks Lon, an ATP dependent protease responsible for degrading unfolded proteins [15]. However, this strain grows at 30°C. This means that any decrease or increase in temperature may result to the death of this cell line. This makes working with this expression system extremely difficult and part of the reasons could be due to the absence of the endogenous molecular chaperones to protect the system from adverse temperatures.

Other protein expression systems such as mammalian and insect cells are very laborious, time-consuming and difficult to maintain [10]. Most pharmaceutically produced proteins such as cytokines [12] and enzymes [4] have previously been produced using Gram-positive expression strains. Bacilli strains are attractive hosts due to their ability to express heterologous protein inside cultured cells and secrete the expressed protein into the extracellular medium [10,15]. Some bacterial strains such as Bacillus brevis (Brevibacillus) choshinensis (Takara-Bio, Japan) have intracellular protease gene (imp) and extracellular protease gene (emp) mutation in order to protect the structural integrity of expressed protein.

According to [14] in living cells newly synthesized and preexisting proteins are at high risk to become misfolded and form aggregation, therefore need molecular chaperones for assistance. Molecular chaperones are proteins that help both new and pre-existing polypeptide to fold properly [14]. Molecular chaperones may act as “holdases” by stabilizing non-native protein conformations from further aggregation or as “foldases” by helping unfolded or partially unfolded proteins to fold to their native state. The expression of heat shock proteins (Hsps) due to heat-stress was first reported by [16] using Drosophila melanogaster cells. Hsps are ubiquitous proteins that are needed to ensure the appropriate folding and conformation of other proteins in the cell [13,17]. Hsps are classified according to their average molecular weight, with major classes consisting of small Hsps (sHsp), Hsp40, Hsp60, Hsp70 and Hsp100 [10,16] (Table 1).

Table 1: Major groups and functions of molecular chaperones.

Due to the significant role molecular chaperones play in maintaining proteome stability, it is therefore not surprising that intracellular Hsps are found in most subcellular compartments such as the nucleus, cytosol, mitochondria and the endoplasmic reticulum (ER) [13,18]. Other molecular chaperones such as Trigger factor, DnaJ/Hsp40, DnaK/Hsp70, GroEL/Hsp60 and GroES/Hsp10 have also been shown to have assisted numerous proteins to fold inside the cellular system [10]. Trigger factor is known to interact with the newly synthesized polypeptide upon its exit from ribosomes [16]. The ribosome associated chaperone Trigger factor is believed to interact with all newly synthesized protein upon their exit from ribosomes [19]. The interaction of these newly synthesized proteins with Trigger factor is to prevent the formation of inclusion bodies because of misfolding of the newly synthesized proteins [13]. However, Trigger factor does not hold onto the substrate for long as the protein continues to be synthesized. Therefore, newly synthesized protein become larger and thus increases the chances for the protein to become misfolded and aggregated. This means Trigger factor could not prevent newly synthesized protein at this stage, and therefore hands the protein over to DnaK/Hsp70 which cooperates with DnaJ/Hsp40 during the folding of the substrate protein [14,18]. Some of the proteins at this stage could have completed their folding processes, therefore do not require further assistance from molecular chaperones. However, other proteins do require further assistance and therefore are being handled by GroEL in combination with GroES to complete the folding process (Figure 2). This work also reports on some of the proteins that have benefited from molecular chaperones system during their folding stages. This includes the features that they probably share in order to be recognized by molecular chaperones and some of the system developed to improve protein production.

Figure 2:Folding pathways of the newly synthesized protein. (A) TF (Trigger factor ) binds emerging nascent chain hand it over (B) Hsp70 homolog DnaK and its cofactors (GrpE, ATP) system, and further hand it over (C) GroEL/Hsp60 chaperonin system for further folding purposes (D) ClpB/Hsp100 is a disaggregating chaperone (Modified from Hartl and Hayer-Hartl, 2002).

Advantages of co-expression with molecular chaperones

The recombinant protein production is required in high quantity, pure and active form for structural studies, however obtaining a pure and active protein has proven to be a challenge. In order to get a required product some of the proteins need to be supplemented with molecular chaperones for proper folding. A number proteins have been successfully produced in pure, soluble and active form, but some proteins could only be produced in either pure but not soluble or active. For example, five different plasmids with 5 different combinations of 6 chaperones molecule transformed into E. coli along with human basic Fibroblast Growth Factor expression plasmid. Each transformant that contain both plasmids for expression of hbFGF and chaperone combinations was induced with proper concentration of related inducers [10]. Subsequently, total amount of produced hbFGF was analyzed based on SDS-PAGE and ELISA. It was indicated that “TF” and “DnaK/DnaJ/GrpE” destabilized hbFGF, while “DnaK/DnaJ/ GrpE/GroEL/GroES” and “GroEL/GroES” combinations were able to stabilize the target protein. It was also revealed that “GroEL/GroES/ TF” combination negatively affected the hbFGF production [10,17]. In addition, individual chaperones were reported to have successfully improved some target proteins. Work done by Stephens and co-workers showed that one of the extensively characterized molecular chaperon, heat shock protein 70 from Plasmodium falciparum improved the production of the target protein from the same origin produced in E. coli [20-22]. Nitrile hydratase (NHase) is an industrially important protein that catalyzes the hydration of nitriles to produce amides. This enzyme has previously been used in the manufacturing of industrial products such as acrylamide and nicotinamide, NHase has also been used in wastewater treatment [10]. Pei and colleagues [8] recently demonstrated that co-expression assays using molecular chaperones such as GroEL/ES, Dnak/J-GrpE and trigger factor significantly promote the soluble expression of recombinant co-type NHase in E. coli. Furthermore, GroEL/ES and Dnak/J-GrpE chaperones-assisted expression systems were shown to produce activated and soluble NHase protein, thus, further reducing the industrial production cost of NHase co-type protein.

Chaperone-assisted expressions of Mab and VLPs

Chaperone-assisted folding expression systems are not only beneficial for industrial applications, but can also be adapted for therapeutic proteins such as antibody and vaccine production. Deuerling and colleagues [18] demonstrated that the co-translation of trigger factor or DnaK with an aggregation-prone antibody efficiently increased the level of production of soluble protein produced. Coexpression with molecular chaperones has also been documented to have improved the solubility and affinity of Single-chain variable fragment (fused Heavy and Light chain) antibodies [11,12,23]. The advantages of chaperone expression have also been documented in the expression of Human papillomavirus 16 E7 oncoprotein using molecular chaperones (DnaK–DnaJ–GrpE, GroEL–GroES) [10]. In the presence of molecular chaperones the expression of soluble proteins is significantly increased in E. coli [14].

The universal system to produce recombinant proteins is still a bottleneck in order to produce large quantities of proteins for structural studies towards designing compounds or drugs for various diseases. Different systems have been proposed to address this challenge. Amongst the challenges is the production of insoluble proteins or some of the proteins are not produced at all because of the system used. E. coli is used as a host system for protein production, due to the presence of its own proteins it is very difficult to bring in foreign proteins in the system. However, molecular chaperones have been studied and are the promising tools to solve this problem [14,24,25]. Their role is to bind to newly synthesized proteins until the fold properly. Molecular chaperones from E. coli and Plasmodium falciparum are well documented, also have been shown to improve some of the recombinant proteins produced in E. coli system [10,22]. However, the question of getting a system that could accommodate all recombinant proteins of different sizes still remains. Therefore, the search for the system that can be used to produce all forms of proteins from different sources is still in progress.

This material is based on the work in progress and is supported financially by the National Research Foundation. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and therefore the NRF does not accept any liability in regard thereto. Financial support is also being acknowledged from the University of Zululand Research Committee. Without their support, this work would not have been started nor finished.