Mouse Interferons: Amino Terminal Amino Acid Sequences of Various Species

Taira, Hideharu; Broeze, Robert J.; Jayaram, B. M.; Lengyel, Péter; Hunkapiller, Michael W.; Hood, Leroy

doi:10.1126/science.7352261

Cited by 106 publications

(19 citation statements)

References 13 publications

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“…Essentially the same sequence was found for human fibroblast interferon produced in the presence of fetal calf serum (unpublished data); identical sequences for the first 13 residues were found for interferon from normal (ref. 1 and this paper) or transformed (Y. H. Tan, personal communication) fibroblasts.…”

Section: Discussionsupporting

confidence: 64%

NH2-terminal amino acid sequence of human fibroblast interferon.

Stein¹,

Kenny²,

Friesen³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met- Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-PheGln-X-Gln-Lys.Other laboratories have reported on the purification and partial structural analysis of human fibroblast interferon (1, 2). We present a novel purification and some additional sequences of human fibroblast interferon. Interferon used in our studies was prepared in serum-free medium and was purified by a procedure based on the combination of affinity chromatography and high-performance liquid chromatography (HPLC).EXPERIMENTAL PROCEDURES Interferon Production and Assay. Crude fibroblast interferon was produced as described by Havell and Vilcek (3), except that serum was omitted from the overnight induction medium. Interferon titers were determined by a cytopathic effect inhibition assay that was modified so that the entire assay could be performed within 16 hr (4). All interferon titers were expressed in terms of reference units/ml, calibrated against the reference standard for human leukocyte interferon (G-023-901-527) provided by the Antiviral Substances Program of the National Institute of Allergy and Infectious Diseases, Bethesda, MD.Purification of Human Fibroblast Interferon on Blue Sepharose. Sodium chloride was added to medium containing interferon to a final concentration of 1 M, and the solution was then pumped onto a 25-ml Blue Sepharose CL-6B (Pharmacia) column (1, 5) at room temperature at a rate of 2.5 ml/min. The unfractionated interferon was maintained on ice during the loading process. The column was washed with 250 ml of sodium phosphate buffer (50 mM Na2HPO4 adjusted to pH 7.2 with HCI) containing 1 M NaCl and 30% (vol/vol) ethylene glycol. The interferon was eluted with the same solution containing 50% (vol/vol) ethylene glycol. Peak fractions of activity were pooled and stored at 40C until used (Fig. 1). Activity appeared to be stable for at least 3 mo at 4VC or in liquid nitrogen.In preparations having a low initial titer, a second passage through Blue Sepharose was required. In these instances, when a total of 25 liters of culture medium containing interferon had been chromatographed, the peak fractions from five columns were pooled and adjusted to 10% (vol/vol) ethylene glycol, 2 M NaCl, and 50 mM Na2HPO4 (pH 7.2). This material was then applied to another Blue Sepharose column. The column was then washed with 250 ml of the sodium phosphate buffer containing 2 M NaCl and 30% ethylene glycol. Some interferon was eluted at this step. The remaining interferon was eluted with the same solution containing 50% ethylene glycol. Interferon that eluted from the second Blue Sepharose column with both 30% and 50% ethylene glycol was satisfactory for use in the next step of the purification. The specific activity of the interferon that was deemed sati...

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Section: Discussionsupporting

confidence: 64%

NH2-terminal amino acid sequence of human fibroblast interferon.

Stein¹,

Kenny²,

Friesen³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…The positions of nine amino acids (positions 2-6, 9-11, and 15, Fig. 2) are identical to those in mouse interferon (5).…”

Section: Resultsmentioning

confidence: 57%

“…Within the past few months, the amino-terminal amino acid sequences of several interferons were reported (3)(4)(5). The major obstacle in determining the sequence of interferon has been the availability of sufficient amounts of pure material.…”

mentioning

confidence: 99%

Amino-terminal amino acid sequence of human leukocyte interferon.

Levy¹,

Shively²,

Rubinstein³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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We reprt the amino-terminal sequence of the first 22 amino acids of human leukocyte interferon. These and other results indicate that human leukocyte interferon consists of many individual species. We, therefore, postulate that diversity in this protein is routinely present and that the human leukocyte interferons represent a multigene family. Although 23 years have passed since the discovery of interferon (1), purification of several interferons has been attained only in the last few years. We previously reported the purification and amino acid composition of human leukocyte interferon (2). Within the past few months, the amino-terminal amino acid sequences of several interferons were reported (3-5). The major obstacle in determining the sequence of interferon has been the availability of sufficient amounts of pure material. Recent advances in manual (6, 7) and automatic (8, 9) sequence analysis have made possible the determination of the primary structure of microgram amounts of protein. We report here the aminoterminal sequence of human leukocyte interferon as determined by automatic and manual sequencing of the native protein and tryptic fragments. In addition, we extend the recently reported amino-terminal sequence and describe two significant differences between our data and those of Zoon et al. (4). EXPERIMENTAL PROCEDURESThe purification of human leukocyte interferon from chronic myelogenous leukemia (CML) cells has been described (2, 10, 11). The sequences of human leukocyte interferon species al and 132 were determined with a modified Beckman spinningcup sequenator. Peptide fragments of a1, a2, and #,-species were produced by digestion with trypsin and purified by high-performance liquid chromatography (HPLC). The sequences of tryptic peptides were determined manually by the Edman reaction (12), and the phenylthiohydantoin derivatives of the amino acids were identified by HPLC (13-15).Automatic Edman degradations were performed in a modified Beckman 890C sequenator on 1.7 nmol of species al of human leukocyte interferon. The modifications, which are similar to those described by and Hun-kapiller and Hood (16), include an improved vacuum system, improved reagent and solvent delivery system, extensive solvent and reagent purification, and a device (17) that automatically converts anilinothiazolinone to phenylthiohydantoin derivatives of amino acids. Proteins are retained in the spinning cup with 6 mg of Polybrene, which, together with 100 nmol of glycylglycine, has been subjected to seven precycles of Edman degradation. Phenylthiohydantoin amino acids were analyzed by HPLC on Du Pont Zorbax octadecylsilica or cyanopropylsilica columns on a Waters Associates chromatograph, by monitoring absorbance at 254 nm and 313 nm. Peak assignments, except for serine, were made by chromatography on a Zorbax octadecylsilica column. Phenylthiohydantoin serine was identified as the "dehydro" derivative on a cyanopropylsilica column. Peaks were integrated and gradient elution was controlled by a Spectra Physic...

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“…MuIFN-a8 has 90 to 95~ nucleotide homology and 80 to 90 9/oo amino acid homology with the sequences of the previously described MuIFN-c~ genes. The deduced amino acid sequence compared with the published Nterminal sequence ofa MuIFN-e protein (Taira et al, 1980) contains five cysteine residues (positions 1, 29, 86, 99 and 139) also present in all M u l F N -~ species identified. The pairs Cysl-Cys99 and Cys29-Cys139 are thought to be involved in disulphide bonds (Wetzel, 1981).…”

Section: Isolation and Characterization Of The Mulfn-~8 Genementioning

confidence: 99%