Purification and structural analysis of interferon

Rubinstein, Menachem

doi:10.1098/rstb.1982.0104

Cited by 6 publications

(1 citation statement)

References 21 publications

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“…The minor residues at positions 1 and 2 could be due to a small amount of initiation at the second methionine at position 3, to incomplete loss of the N-terminal methionine, or to other E. coli proteins present in the interferon preparation. At step 1 of the degradation about 5 nmol of threonine was released, and since the number of antiviral units was known to be 5 x 106, this gave a figure for the specific activity of 0.5 x 108 International Units (IU)/mg protein, in reasonable agreement with the previous figure of 2 × 108 units/mg protein for this material (Slocombe et al, 1982), which was similar to that reported for unmodified IFN-az of 3 x l0 s IU/mg protein (Rubinstein, 1982).…”

Section: Quantification Of Phenylthiohydantoin (Pth)-amino Acids Idensupporting

confidence: 80%

Characterization and Properties of a Modified Human Interferon- -Containing an Additional 18 Amino Acids at the N-Terminus

King

Burke

Northrop

et al. 1983

Journal of General Virology

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SUMMARYA modified human interferon-or 2 was produced in Escherichia coli cells infected with phage M13 mp7 containing an interferon-~ gene. After purification by immunochromatography with the monoclonal antibody NK2, the N-terminal amino acid sequence was determined. The N-terminal methionine was absent but an additional sequence of 18 amino acids at the N-terminus was retained. The modified interferon-or2 was indistinguishable from authentic interferon-~2 in its ability to activate natural killer cells, to slow the growth of Daudi cells, and to confer virus resistance on heterologous cells.We have recently reported the use of phage MI3 mp7 as a vector for high level expression of an interferon-ct z gene (Slocombe et al., 1982). The predicted amino acid sequence of the recombinant interferon contained an additional 19 amino acids at the N-terminus, although this could not be confirmed by direct determination of the N-terminal amino acid by the dansylation method. The recombinant interferon was recognized by the monoclonal antibody to interferon-~, NK2, and had a similar specific activity as assessed by the immunoradiometric assay (Secher & Burke, 1980;Secher, 1981). It also showed cross-species antiviral activity similar to a mixture of interferon-~ species. It seemed, therefore, that either the additional amino acids at the Nterminus had no effect on antiviral activity or that the recombinant product had been processed by proteolytic cleavage to remove the additional amino acids. We now report that only the Nterminal methionine residue is removed, and that the additional 18 amino acids at the Nterminus have no effect on several biological activities of interferon-~2.The bacterial interferon-c~2 was prepared as described by Slocombe et al. (1982) except that double-strength YT broth was used, since on average threefold more interferon was produced. Variations in a number of other parameters [carbon source, density of the bacterial culture, multiplicity of infection and concentration of isopropyl-fl-D-thiogalactopyranoside (IPTG)] had little effect on the interferon yield. Indeed, cultures incubated in the absence of IPTG produced interferon from about 2-5 h after infection, the final yield being 10 to 25~ of that obtained from induced cultures, suggesting that the replication of the phage may titrate out the lac repressor produced by the bacterial chromosome.When the crude product from the bacterial cultures was purified on NK2-Sepharose as described by Slocombe et al. (1982), and analysed by polyacrylamide gel electrophoresis, the major component had a mol. wt. of 20 500. However, there were also small amounts of some proteins of higher molecular weight in the gel. These were shown to be bacterial products by use of the M 13 mp7-ct2 clone designated W26 (Slocombe et aL, 1982) in which the orientation of the interferon gene is reversed, so that no interferon is produced. Purification of the lysate from a W26-infected culture by immunochromatography on NK2-Sepharose showed that these minor bands were still presen...

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Section: Quantification Of Phenylthiohydantoin (Pth)-amino Acids Idensupporting

confidence: 80%