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A ribonuclease (RNase) has been isolated from normal human pancreas obtained upon autopsy. About 5 mg of RNase is normally recovered per kilogram of pancreas, equivalent to ca. 70% of the total activity and a 700-fold purification from the initial acidified extract. The specific activity of the purified enzyme is identical with that of bovine pancreatic ribonuclease, and a single component is found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and reversed-phase high-pressure liquid chromatography. Aggregation of the protein is found upon ultracentrifugation under native and denaturing conditions, and several bands of equal specific activity are seen in polyacrylamide gel electrophoresis of the native protein. At least two components are glycoproteins. A molecular weight of 15 000 is estimated from sodium dodecyl sulfate gel electrophoresis, gel filtration, and amino acid and peptide analyses. The enzyme is related to bovine pancreatic RNase, but distinguishable by amino acid analysis, tryptic peptide maps, and low cross-reactivity of antibodies with the heterologous enzymes. The human enzyme is also inactivated by treatment with iodoacetic acid at pH 5.5 and is essentially identical with bovine RNase in its far-ultraviolet circular dichroism spectrum. The human RNase is like bovine pancreatic RNase catalytically; RNA is cleaved at pyrimidine residues, and activity against poly(cytidylic acid) is high.

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Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.