Identification and Cloning of the Recombinant Human IFNa Species

Because recombinant DNA technology offered an opportunity to produce large amounts of Hu-IFNs economically, many scientific teams set out to clone them in bacteria. Several groups isolated recombinants for several Hu-IFN-a species (Maeda et al. 1980; Nagata et al. 1980) and for IFN-P (Derynck et al. 1980; Goeddel et al. 1980a; Houghton et al. 1980; Maeda et al. 1980; Taniguchi et al. 1980), obtaining the clones by somewhat different but analogous approaches. The cloning and expression of Hu-IFN-aA (Hu-IFN-a2a) as an illustration of these procedures is described.

Isolating Hu-IFN DNA sequences was a formidable task since it meant preparing DNA recombinants from cellular mRNA that was present at a low level. This task had never been accomplished previously from a protein whose structure was unknown. In addition, in order to reconstruct DNA recombinants which would express natural IFN, it is useful to know the partial amino acid sequence of the proteins, particularly at the NH2- and COOH-terminal ends. Without this information, synthesis of natural Hu-IFN in bacterial cells would not have been definitive. Thus, purification of the Hu-IFNs and determination of their structure (Allen and Fantes 1980; Hobbs et al. 1981; Hobbs and Pestka 1982; Knight et al. 1980; Levy et al. 1981; Rubinstein et al. 1978a, 1979c, 1981; Shively et al. 1982; Zoon et al. 1979) assisted us in these efforts.

To isolate recombinants containing the human DNA corresponding to IFN-a, we developed a number of procedures. First, it was necessary to isolate and measure the IFN mRNA. This was accomplished several years earlier when IFN mRNA was translated in cell-free extracts (Pestka et al. 1975; Thang et al. 1975) and in frog oocytes (Cavalieri et al. 1977a; Cavalieri et al. 1977b; Cavalieri and Pestka 1977; Reynolds et al. 1975). The next step was to prepare sufficient mRNA from cells synthesizing IFN, and this was accomplished with both fibro-blasts and leukocytes (Familletti et al. 1981a; McCandliss et al. 1981a). A library of complementary DNA (cDNA) was prepared from a template of partially purified mRNA isolated from human leukocytes synthesizing IFN. Next was to find in this vast library of recombinant plasmids those which contained DNA encoding IFN. We devised an indirect two-stage procedure to identify clones containing interferon sequences. In the first stage, we screened all the bacterial colonies to find those with cDNA made from the RNA of induced cells; among these there might have been some carrying IFN cDNA. We therefore screened all the recombinants for their ability to bind to mRNA from cells synthesizing IFN (induced cells), but not to mRNA from uninduced cells (those not producing IFN). To do this, individual transformed colonies were screened by colony hybridization for the presence of induced specific sequences with 32P-labeled IFN mRNA (mRNA from induced cells) as probe. In the presence of excess mRNA from uninduced cells, recombinants that were representative of mRNA sequences existing only in induced cells should be evident on hybridization. This screening procedure allowed us to discard about 90% of the colonies: since their plasmids carried no induced cDNA, these could not encode IFN (Maeda et al. 1980, 1981).

In the second stage, we identified those recombinants containing the IFN DNA sequences among the remaining 10%. To accomplish this, we pooled the recombinant plasmids in groups of ten and examined these for the presence of IFN-specific sequences by an assay that depends upon hybridization of IFN mRNA to plasmid DNA (Maeda et al. 1980; McCandliss et al. 1981b). Plasmid DNA from ten recombinants was isolated and covalently bound to diazoben-zloxymethyl (DBM) paper. The mRNA from induced cells was hybridized to each filter. Unhybridized mRNA was removed by washing. After the specifically hybridized mRNA was eluted, both fractions were translated in Xenopus laevis oocytes. Once a positive group had been found (one in which the specifically hybridized mRNA yielded IFN after microinjection into frog oocytes), it was necessary to identify the specific clone or clones containing IFN cDNA. The individual colonies were grown, the plasmid DNAs were prepared, and each individual DNA was examined by mRNA hybridization as above. By these procedures, a recombinant, plasmid 104 (p104), containing most of the coding sequence for a Hu-IFN-a, was identified (Maeda et al. 1980). The DNA sequence was determined and found to correspond to what was then known of the amino acid sequence of purified Hu-IFN-a (Levy et al. 1980; Levy et al. 1981). The cDNA insert in plasmid p104 contained the sequence corresponding to more than 80% of the amino acids in IFN-aA, but not for those at its amino-terminal end. It was, therefore, used as a probe for finding a full-length copy of the IFN cDNA sequence that could be used for expression of Hu-IFN-aA in E. coli (Goeddel et al. 1980b). In addition, p104 DNA was used to isolate DNA sequences corresponding to other IFN-a species directly from a human gene bank.

Examination of the coding regions of the IFN-a genes that have been isolated in our laboratory and others have shown that these correspond to a family of homologous proteins (Pestka 1983a; Rubinstein et al. 1979c) that are closely related to each (Table 2). Thus, the previously discovered heterogeneity in Hu-IFN-a was at least in part the result of distinct genes representing various expressed Hu-IFN-a sequences. The cloned Hu-IFN-aA (Hu-IFN-a2a), the first one we isolated, corresponds to one of the natural Hu-IFN-as that we purified by HPLC. By procedures similar to those described for plasmid p104, plasmid p101 was shown to contain the sequence for Hu-IFN-p. Thus, the nucleotide sequences coding for Hu-IFN-a and Hu-IFN-P were identified.

Was this article helpful?

0 0

Post a comment