Christine Citti*, Marion Brank and Renate Rosengarten
4. Generation of Recombinant Antigens
4.1 Basic Principles
The basis for the production of a recombinant protein is the expression of an open reading frame into prokaryotic or eukaryotic heterologous systems. Using standard molecular procedures, heterologous genes are introduced into an expression vector under the transcriptional control of a promoter. Because the overexpression of foreign proteins might be detrimental to the host cells at an early stage of the multiplication, most systems use promoters that are activated on demand by addition of an inducer (see for illustration Fig.
3 B). This is for instance the case of the bacterial promoters Ptacor T7 that respectively require a source of isopropyl β-D-thiogalactopyranoside (IPTG) or T7 RNA polymerase to be functional. In addition to an inducible promoter, these expression vectors also provide a ribosome binding site and a start codon which are localized downstream of the promoter and upstream of a multiple cloning site (MCS) for the in-frame insertion of the sequence to be expressed. A number of vectors additionally offers a variety of tags adjacent to the MCS which purpose is to facilitate the purification and the detection of the expressed protein as illustrated in Fig. 1.
Most tags are linked to the N-terminal portion of the protein of interest, but some vectors offer the alternative of fusing the tag to the C-terminal part of the protein. Furthermore these tags contain a region overlapping the MCS and encoding a cleavage motif for releasing the product of interest. Finally, some expression vectors are equipped with a sequence signal located upstream of the tag for periplasmic localization.
4.2 Prokaryotic versus Eukaryotic Expression Systems
In the past few years, the number of expression systems commercially available for the production of recombinant proteins suitable for use in bacteria or in various eukaryotic cells has constantly increased. The most documented are (i) recombinant E. coli cells, (ii) recombinant yeast cells, (iii) a baculovirus-driven insect cell expression system (Kost and Condreay, 1999), (iv) recombinant vaccinia viruses for the transient expression of foreign proteins into various mammalian cells (Fuerst et al., 1986) and (v) mammalian chinese hamster ovary cells for continuous expression of recombinant proteins (Collen et al., 1984).
The problem in using bacterial systems is that they do not support most of the post-translational modifications achieved by eukaryotic systems and are therefore not suitable for the expression of eukaryotic or viral proteins that may require specific glycosylation, acylation or phosphorylation for displaying their antigenic properties. Overall, the choice of the expression system mainly depends on the nature of the protein to be expressed, and to whether modifications or secondary structures are of antigenic relevance for the assay.
The pro’s and con’s of these systems and detailed protocols for expression of foreign antigens along with tips for problem solving have been extensively reviewed (Reschl, 1998).
Christine Citti, Marion Brank and Renate Rosengarten
Christine Citti, Marion Brank and Renate Rosengarten
Figure 1. Outline illustrating the successive steps to be performed for the purification of a recombinant fusion protein. The gene of interest (shaded open arrow) is cloned into an expression vector, in frame with a sequence encoding a tag (hatched open arrow). Depending whether the gene is inserted downstream or upstream of the tag-encoding sequence, the recombinant product will be fused to the tag at its N-terminal or C-terminal end. Following transformation of the host cells, the expression of the fusion protein will be induced by adding a factor that will activate the transcription of the inducible promoter (Pi). Shaded circles: protein of interest; hatched circles: tag; black circles: proteins from the host cells; vertical shaded rectangle: affinity column.
4.3 Production of Recombinant Fusion Proteins
As mentioned above, a wide range of expression vectors are commercially available for the production of recombinant proteins. Most bacterial expression vectors either use the T7 promoter or the Ptac in conjunction with a polyhistidine tag which can bind to nickle-nitrilotriacetic acid (Ni-NTA) columns, or with naturally occurring proteins which include (i) the maltose binding protein (MBP), a protein which has an affinity to amylose, (ii) the gluthatione S-transferase (GST) which has an affinity to gluthatione, and (iii) protein A which has an affinity to immunoglobulin. The eukaryotic expression system based on the pFASTBACHT series of bacculovirus shuttle vectors (GIBCO BRL) also express polyhistidine-tagged proteins.
Since the factors leading to high-level expression of the protein of interest in foreign hosts are usually not known, the choice of which affinity tags to use for the expression of a particular protein is empirical. Following purification, the fusion protein can be directly used, but in most of the cases the removal of the affinity tag is necessary either for proper folding of the protein of interest or for avoiding false positive serological reactions due to the presence of antibodies directed to the tag in the sera to be tested. Because removal of the tag using chemical procedures is often difficult to control, it is mainly performed by enzymatic cleavage using proteases, such as the enterokinase, the thrombin or the factor Xa. Table 2 presents few systems commercially available for the expression of recombinant proteins in E. coli or in mammalian cells.
As mentioned above, the factors influencing the level of expression of a particular recombinant protein in a prokaryotic or eukaryotic host system are usually unknown.
However, a number of relevant problems often encountered with the expression of recombinant proteins can be overcome. These include solubilization, toxicity and proper refolding of the recombinant proteins. As well, the poor expression of some mycobacterial genes in E. coli partly result from their content in low-usage E. coli codons. Codon replacement by site-directed mutagenesis has been recently shown to enhance the production of recombinant Mycobacterium tuberculosis antigen 85A and superoxide Christine Citti, Marion Brank and Renate Rosengarten
Table 2. Most Commonly Used and Commercially Available Expression Systems.
Affinity tag Affinity column Elution ligand Supplier Vector1
designation
Glutathione S-transferase Gluthatione sepharose Reduced gluthatione Pharmacia pGEX
6Xhis Ni-NTA2 Imidazole Quiagen, pQE
Novagen pET
GibcoBRL pFASTBACHT
Maltose Binding Protein Amylose resine Maltose New England Biolabs pMAL
Protein A IgG Binding IgG sepharose IgG Pharmacia pEZZ 18
Domain pRIT2T
1and derivates 2Nickel-nitrilotriacetic acid
dismutase in E. coli indicating that for some bacterial genes this strategy might be applicable for obtaining overexpression in E. coli (Lakey et al., 2000). In the case of the recombinant antigen 85A, an urea-based resolubilisation protocol similar to methods used in refolding of other recombinant proteins was also necessary to obtain the soluble recombinant antigen.