Cloning Allergens from A. fumigatus:
Because of the above mentioned problems associated with antigen purification and standardization, newer approaches have been investigated. This has resulted in the use of molecular cloning and expression of allergens. A wide variety of techniques have been used to clone complementary DNA (cDNA) encoding the allergens. Young growing cultures in the logarithmic phase, usually 72-96 hours old cultures have been used to extract the RNA. The total RNA from the washed mycelium was extracted with guanidium isothiocynate (Chirgwin et al., 1979) and messenger RNA (mRNA) purified using oligo dT cellulose affinity chromatography (Aviv and Leder, 1972). Reverse-transcribed mRNA containing appropriate restriction sites has been used to construct expression libraries in the lambdaZAP II vector (Kumar et al., 1993b; Moser et al., 1992,).
However, efficient handling of large libraries is greatly facilitated if screening can be based on selective enrichment of clones expressing genes of interest (Crameri and Suter, 1993). Starting from cDNA, the fast and selective way to clone a gene is by amplification of the desired DNA fragment by polymerase chain reaction (PCR) (McPherson et al., 1993). PCR has been successfully used to clone allergens and allergen isoforms (Breiteneder et al., 1993; Larsen et al., 1992), including Asp f 1 (Moser et al., 1993). A major limitation of this technology is the requirement of sequence information particularly from the N-terminal part of the protein, in order to be able to design primers for the PCR amplification reaction. The most frequently used strategy to identify IgE-binding molecules in cDNA expression libraries is based on screening of the solid phase--immobilized libraries (Mierendorf et al., 1987) with IgE-containing sera from allergic patients or with antibodies raised against purified allergens (Achatz et al., 1995; Banerjee et al., 1996; Kumar et al., 1993b; Moser et al., 1992). Obviously, immobilization of a library hampers selective enrichment of clones expressing proteins of interest by specific gene product/ligand interaction (Crameri et al., 1994).
A rational approach to clone allergens and clone cDNAs would be to selectively enrich clones expressing products of interest from cDNA expression libraries. Such an approach requires, however, a physical linkage between gene product and genetic information encoding the gene (Crameri and Suter, 1993; Crameri et al., 1994). This basic requirement is not fulfilled by lambda-based cDNA libraries where genetic information is integrated into the phage genome, and gene product expression exploiting the machinery of the host cell during the lytic process, are not physically linked (Sambrook et al., 1989). In contrast, filamentous phage engineered to display gene products on their coats, contain the DNA encoding the displayed gene product as a part of their single-stranded genome (Barbas and Lerner, 1999; Smith, 1985) resulting in two immediate consequences. First it allows selective isolation and amplification of a particular phage encoding a desired gene product from pools of millions of phages. Selection is accomplished by interaction between the displayed gene product and a ligand immobilized on a solid phase, and the selected phage are amplified by infection of E. coli cells which, after helper rescue, produce a large number of new phage (Smith, 1985). Second, the amino acid sequence of a selected gene product can be readily elucidated by sequencing of the DNA of the relevant section of the phage genome. These procedures allow easy enrichment of phages displaying a gene product with a particular character of interest using the power of affinity-based selection, and are therefore, more versatile than classical cloning systems (Appenzeller et al., 2000; Crameri et al., 1996c; Kay et al., 1996; Smith, 1985; Smith and Scott, 1993). However, one of the limitations of filamentous phage as display vectors for cDNA libraries is a direct consequence of the capsid structure. Since the integrity of the carboxy-terminus of pIII is essential for efficient phage assembly (Crameri, 1997), insertions of foreign proteins can only be tolerated at the amino terminus. cDNAs derived from poly A+-selected mRNA, however, cannot be assembled in the phage coat by this approach since the translation stop codon present at the 5' end of the cDNA prevents the synthesis of pIII fusion proteins. To overcome this limitation, an indirect fusion strategy was devised where cDNA inserts fused to the 3' end of the Fos gene are co-expressed with a gene encoding a Jun leucine zipper-pIII fusion (Crameri and Suter, 1993). The high affinity interaction between the Jun and Fos leucine zippers efficiently link the cDNA products to the phage capsid (Barbas and Lerner, 1999; Crameri et al., 1994), allowing screening of cDNA libraries in liquid phase (Crameri et al., 2000a; Crameri and Walter, 1999b).
Compared with the screening of lambda libraries, the screening procedure for cDNA libraries displayed on phage surfaces have manifold advantages. The most important one results from the fact that the phage library is kept in liquid phase, where only phages with affinity to the ligand are retained on the surface after washing (Crameri, 1997; Crameri et al., 1994). Successive rounds of phage growth and selection allow enrichment of phage-displaying proteins with affinity for the ligand and the system has been successfully used to clone allergens from different complex allergenic sources (Crameri et al., 1994; Eriksson et al., 2000; Kleber-Janke et al., 1999; Lindborg et al., 1999). Many A. fumigatus allergens have been cloned using both, classical cloning systems and phage surface display and will be discussed in the following section.