Purification to homogeneity and partial characterization of interleukin 2 from a human T-cell leukemia.

Abstract

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A rmal purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10' units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.Interleukin 2 (IL-2), formerly known as T-cell growth factor, was first described as a lymphokine capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific effector T lymphocytes (1, 2). IL-2 partially purified from mouse (3, 4), rat (5, 6), and human (7, 8) normal T lymphocytes has been found to retain different types of biological activity, including: (i) marked enhancement of thymocyte mitogenesis (7, 9); (ii) promotion of long-term in vitro proliferation of antigen-specific helper or killer T-cell lines (3, 10); and (iii) induction of cytotoxic Tlymphocyte reactivity and plaque-forming cell responses in cultures of nude mouse spleen cells (7,9). These activities indicate that IL-2 may be useful in augmenting immune responses and restoring immune-deficient T-cell populations (nude mouse spleen cells) to normal levels of cellular and humoral immunity. Furthermore, these results suggest that IL-2 production and response are important parameters of immunological functions and their measurement may be useful in clinical diagnosis of aberrant immune status.The sources and techniques that have been used to produce IL-2 have yielded low concentrations of IL-2, with large volumes of conditioned media being required to obtain only small quantities of human IL-2 activity. As a consequence, sufficient quantities of concentrated human IL-2 have not been available for in vivo experiments or for its characterization. Several groups have reported purification procedures for both murine (9, 11) and human (7,(12)(13)(14) IL-2, although the purity of these preparations is not clear. The present study reports a procedure for the purification of human IL-2 to homogeneity using several steps of reversed-phase high-performance liquid chromatography (HPLC). Chemical characterization of the purified lymphokine is reported. EXPERIMENTAL PROCEDURESSource of IL-2. Human leukemia T cells designated Jurkat-H33HJ-JAI (a subclone of the parent Jurkat-FHC1C cell line) (15) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units of penicillin per ml, 50 ug of gentamycin per ml, and 300 .g of fresh L-glutamine per ml. The cells were grown in T175-cm2 flasks (volume, 150 ml) in a humi...

Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.

“…Beginning with an asparagine codon at position 19 there are 9 contiguous amino acids, which are identical in human IL-2. This sequence contains an alanine residue, which is the NH2-terminal amino acid deduced for mature human IL-2 (11,25). It is likely, therefore, that the mature form of the secreted mouse IL-2 begins with an alanine residue and the preceding 20 amino acids constitute the putative leader sequence that is removed by proteolytic processing.…”

Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines.

“…A mouse IL-2 cDNA was cloned from a library prepared from LBRM-33 lymphoma cell mRNA by Immunex Corporation and was expressed in yeast using the yeast a factor promoter and leader sequences to direct synthesis and secretion (16). rIL-2 was purified to homogeneity as previously described (17), and had a specific activity of 10 ~ U/rag. A mouse IL-3 cDNA was also cloned from the above-detailed library by Immunex Corporation, expressed in yeast, and resultant rIL-3 was purified to homogeneity (18).…”

Structures of the major carbohydrates of natural human interleukin‐2

“…Recently, procedures for the purification of human IL-2 from various cell sources have been published [5][6][7][8]. Taniguchi and coworkers reported the first cloning and nucleotide sequence of a cDNA coding for 1L-2 from the human leukemic T cell line Jurkat [9].…”