Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs

Abstract

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5′ and 3′ regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5′ end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5′ region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3′ end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3′ fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5′ and 3′ ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea – sodium dodecyl sulphate –polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-β-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small. These studies demonstrate the feasibility of producing mature human SP-B through recombinant DNA technology, but indicate that it may be necessary to use a fusion protein approach to obtain sufficient amounts for the formation of artificial pulmonary surfactant.Key words: pulmonary surfactant (human), hydrophobic proteins, artificial pulmonary surfactant, in vitro translation.