Stanley Stein

Characterization of recombinant human interleukin-2 with micromethods

Highly purified recombinant human interleukin-2, expressed in Escherichia coli, was analyzed by micromethods. N-Terminal sequence analysis showed that methionine at position 0 was found in 90% of the molecules and not completely removed in post-ribosomal processing. A complete peptide map of the reduced and S-carboxymethylated protein was obtained by high-performance liquid chromatography after tryptic digestion, and the fragments were identified by amino acid analysis and automated Edman sequence analysis. Using a double-label S-carboxymethylation procedure, it was determined that there is a disulfide linkage between the cysteine residues at positions 58 and 105. The third cysteine residue at position 125 was found to be present as the free sulfhydryl.

The primary structure of monkey pituitary growth hormone.

Monkey growth hormone (MGH) has been purified by reverse-phase high-performance liquid chromatography (HPLC). Tryptic digests of MGH were separated by HPLC and paper electrophoresis. From amino acid composition, from NH2-terminal residue and sequence analyses of these tryptic peptides, and from their alignment with those of human growth hormone, the primary structure of MGH was proposed. There are only four residues which are different in these two growth hormone molecules.

Use of high-performance liquid chromatography for preparing samples for microsequencing

A method has been developed for the microsequencing of protein at subnanomole levels. The protein is carboxymethylated and freed of salts and reagents by reversed-phase chromatography prior to automated Edman degradation on a gas-phase sequencer. The carboxymethylated protein can also be fragmented chemically or enzymatically for further sequence analysis. The analytical techniques used to monitor the progress of the reactions all have picomole level sensitivity.

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

Abstract The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH 2 -terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.

Isolation of proteins from crude mixtures with silica and silica-based adsorbents.

Silica and silica-based adsorbents have been used to isolate specific proteins from crude mixtures. Acid-activated Nugel silica and Nugel SP-Silica were used to extract human immune interferon from the conditioned medium of mixed lymphocyte cultures. Human interleukin-2 was extracted from the conditioned medium of Jurkat cells with LiChroprep RP-8. In each case, batch absorption was accomplished by gentle stirring in a microcarrier vessel. The adsorbate was collected by sedimentation, and either batch-eluted or packed into a column for gradient elution. A high degree of concentration and purification was achieved with a high recovery of biological activity.

Characterization of small amounts of peptides and proteins

The use of microchemical methods for the characterization of both natural and recombinant proteins of biomedical importance is discussed. The methods include gel electrophoresis, high-performance liquid chromatography, protein fragmentation, amino acid analysis and automated Edman degradation. Each procedure is applicable at the picomole level.