Leo S. Lin

Production, partial purification and characterization of human and murine interferons—Type II

Abstract Human and murine type II interferons were produced from mitogen stimulated lymphocyte suspensions. Priming of human lymphocytes with type I human interferon prior to induction slightly increased type II interferon production, but priming of mouse spleen lymphocytes with type I mouse interferon had no apparent effect on type II interferon production. Incorporation of β-mercaptoethanol into culture medium enhanced production of mouse type II interferon but had no effect on human type II interferon yields. Both human and murine type II interferons could be purified from crude culture medium by chromatography on polyribouridylic acid (poly U)-agarose by a method identical to that used for purification of type I interferons, to specific activities of about 104.5 U/mg protein. Both interferons could also be purified from culture medium by chromatography on controlled-pore glass by modifications of methods used for purification of type I interferons, giving specific activities of about 104.0 U/mg protein. When type II interferons were purified by sequential chromatography on controlled-pore glass followed by poly U-agarose, specific activities of approximately 106 units of type II interferon/mg protein were obtained. These partially purified type II human and murine interferons were compared to type I human and murine interferons purified by similar methods to similar specific activities, and each could be distinguished by their antigenicities, acid labilities and host cell specificities.

Characterization of the heterogeneous molecules of human interferons: differences in the cross-species antiviral activities of various molecular populations in human leukocyte interferons.

Summary Human leukocyte interferon (HuLeIF) preparations were separated into populations of molecules with different sizes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and with different charges, by isoelectric focusing. These populations with different sizes and charges were analysed for their antiviral activity on homologous cells and on heterologous (bovine) cells. The distribution of interferon activity into two broad peaks by SDS-PAGE was similar whether assayed on human or bovine cells. However, within these peaks, the relative ratio of the activity in human cells and bovine cells varied significantly: while most of the size components had similar human/bovine cell activities, the fastest migrating component (apparent mol. wt. ∼ 13500) was more than 100 times more active on bovine cells than on human cells. The peaks of activity in isoelectric focusing were distributed from pH 5.5 to 7.0. There was generally correspondence between human and bovine cell activities, but while the more neutral pH range peaks were consistently slightly more active on human cells than on bovine cells, the more acid range peaks were always slightly more active on bovine cells than on human cells. However, with the most acidic peak, there was more than 100 times greater activity on bovine cells than on human cells. These data show that the heterogeneity of HuLeIFs is greater than merely two size populations, and data confirm that different forms of human leukocyte interferon can vary markedly in biological activity.

[26] Purification of recombinant human interferon β expressed in Escherichia coli

Publisher Summary This chapter discusses the purification of recombinant human interferon β (IFN-β) expressed in Escherichia coli . Many purification methods developed for native (glycosylated) human IFN-β are not applicable to nonglycosylated recombinant IFN-β obtained after expression in Escherichia coli . A method is developed for large-scale purification of recombinant IFN-β and IFN-β. IFN-β is a highly active and stable form of IFN-β containing a cysteine to serine substitution at position 17 introduced by in vitro site-specific mutagenesis. The interaction of sodium dodecyl sulfate (SDS) and IFN-β has been utilized in the purification scheme presented. Recombinant human interferons have been expressed in mammalian, insect, yeast, and bacterial cells. A method for the purification of recombinant human IFN-β and an active variant, IFN-β, obtained through genetic engineering, is described. Both proteins are nonglycosylated, lack the amino-terminal methionine present in the native protein and occur as aggregates when expressed at high levels of E. coli . Aggregation is presumably a consequence of the intrinsic hydrophobicity of IFN-β. Detergents, such as sodium dodecyl sulfate or sarkosyl, and chaotropic agents, such as guanidine hydrochloride, effectively solubilize the biological activity present in E. coli cells. This observation and the fact that IFN-β at neutral pH is poorly soluble in the absence of ionic detergents, led to a purification scheme based on the interaction of IFN-β with dodecyl sulfate.

Antiviral activities of interferons

Interferons are a group of proteins produced by eucaryotic cells when they are stimulated by virus infection, double-stranded RNA and various other inducers. Cells, when treated with interferons, exhibit a number of responses, the principal action of interest in this review being the establishment of virus resistance. There are however a number of other responses that have been described, such as inhibition of non-viral agents; priming; blocking; cell-multiplication-inhibition; toxicity enhancement; enhanced synthetic activities; surface alteration; enhanced immunolysis; enhanced phagocytosis and various immune modulations. In this survey we will first discuss the antiviral action of interferon both in vitro and in vivo and describe the non-antiviral activities, as these are likely to contribute to virus resistance mechanisms in the animal.

Differential inactivation and separation of homologous and heterologous antiviral activity of human leukocyte interferon by a proteolytic enzyme.

Abstract Human leukocyte interferon (HuLeIF) can express its antiviral activity on both human and bovine cells. The rates of inactivation of HuLeIF by α-chymotrypsin, as expressed on human and bovine cells, are not the same: the ability to induce activity on human cells is lost significantly more rapidly than the activity detected on bovine cells; usually a margin of greater than one hundred-fold exists after α-chymotrypsin treatment. HuLeIF, when subjected to analysis on 10% SDS-PAGE, can be separated into two molecular weight species, one having apparent molecular weight of approximately 21,000 daltons, the other 18,000 daltons. A more rapidly migrating form (molecular weight 16,500 daltons) can also be isolated, which is considerably more active on bovine cells than on human cells. α-chymotrypsin-treated samples analyzed by SDS-PAGE show a clear separation of the activities expressed on human and bovine cells. The residual activity detected on human cells is isolated only in the 21,000 component while the activity found on bovine cells is recovered only as the 16,500 dalton species.

[69] Purification of human leukocyte interferon by two-dimensional polyacrylamide gel electrophoresis

Publisher Summary The homogeneous human leukocyte interferon can be obtained by devising a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique. A modified two-dimensional gel electrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications made are (1) the omission of reducing agents, such as β-mercaptoethanol, in all solutions and (2), the increased length of the isoelectric focusing gel from l I cm to 24 cm with the corresponding increase of the second-dimension sodium dodecyl sulfate (SDS) gel slab to 35 cm wide by 30 cm in height. Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 10 9 units per milligram of protein. This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios.

Characterization of the size and charge heterogeneities of human leukocyte interferon populations

SummaryHuman leukocyte interferons are separable into two size components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and are separable into two charge-components by DEAE-BioGel A chromatography. However, each of the charge-components resolved by ion-exchange chromatography contained both size-components, when analysed by SDS-PAGE. Thus, there are more than two distinct molecular populations of human leukocyte interferons.

Characteristics of spontaneously produced and of virus-induced human LuKII cell interferons

SummarySeveral human lymphoblastoid cell lines produced significant levels of interferon (IFN) activity in the absence of any IFN inducees. We have partially purified spontaneous IFN (SpIFN) from LuKII cells and compared it with the IFN activity produced by the same cells following Sendai virus induction. Virus-induced LuKII IFN reached maximum titers of 1000–6000 reference units/ml at 12 to 15 houts post-induction and produced a heterogeneous electrophoretic profile with major (18,500 dalton) and minor (23,500 dalton) species. Spontaneous LuKII IFN was produced very slowly over several days and reached maximum titers of 100–1000 reference units/ml. Crude SpIFN (25–100 ref. units/ml) was purified to 1–6×105 ref. units/ml with a 53–80 percent overall recovery, and it consistently migrated as a homogeneous 20,000 dalton band upon SDS gel electrophoresis. Although spontaneously produced and virus-induced lymphoblastoid IFNs differed in their ability to be neutralized by anti-mouse IFN antiserum (18), both types of IFN were only 1 percent cross-reactive on heterologous mouse L cells. We conclude that IFN definitely can be produced by many human lymphoblastoid cell lines in the absence of inducers, and that LuKII SpIFN has different characteristics than the IFNs produced by the same cell line following viral induction. Since it is now known that there are many IFN-α genes (3, 10), it seems likely that these cell lines may provide a system for studying the selective expression of one or more of these genes.

Purification and properties of native and modified interferons.

The variety in the interferon forms produced by cells of a given animal species is likely to be meaningful to the organisms producing these molecules, but this heterogeneity of interferon molecules reveals itself to us only as a curious phenomenon. To date the only functional distinction apparent in these diverse interferon forms is their pharmacological properties.’ The heterogeneity of mouse type I interferons exhibited as size components of 38,000 and 22,000 daltons in SDS-polyacrylamide appears to arise from glycosylation of precursor polypeptide, the 16,000 dalton “ in te r fe r~ id .”~ As yet we have been unable to discern any functional differences in these apparently extensively glycosylated, partially glycosylated. or nonglycosylated mouse interferon forms, as each appears equally able to induce antiviral, priming, toxicity-enhancing, and cell-multiplication-inhibitory activities5 Similar studies with human leukocyte interferon have suggested that the size heterogeneities of these molecules, revealed as 21.000 and 15,000 dalton components in SDS-polyacrylamide gels,’ is also carbohydrate-contributed. Thus, periodate oxidation eliminates the size and charge heterogeneity of human leukocyte interferons, apparently converting the larger forms to the smaller molecule^,^ and human leukocyte interferon preparations produced in the presence of glycosylation inhibitors contain only the smaller interferon forms: Our recent studies have been directed toward identification of the origins of the heterogeneities of human leukocyte interferons, by attempting conversion of one form to another, either by glycolytic or proteolytic processing, and by identification of the primary translation product of the interferon messenger RNAs obtained from different lymphoid cell populations producing distinct forms of human leukocyte interferons.

Current Molecular-Targeted Therapies in NSCLC and Their Mechanism of Resistance

Lung cancer is treated with many conventional therapies, such as surgery, radiation, and chemotherapy. However, these therapies have multiple undesirable side effects. To bypass the side effects elicited by these conventional treatments, molecularly-targeted therapies are currently in use or under development. Current molecularly-targeted therapies effectively target specific biomarkers, which are commonly overexpressed in lung cancers and can cause increased tumorigenicity. Unfortunately, several molecularly-targeted therapies are associated with initial dramatic responses followed by acquired resistance due to spontaneous mutations or activation of signaling pathways. Acquired resistance to molecularly targeted therapies presents a major clinical challenge in the treatment of lung cancer. Therefore, to address this clinical challenge and to improve lung cancer patient prognosis, we need to understand the mechanism of acquired resistance to current therapies and develop additional novel therapies. This review concentrates on various lung cancer biomarkers, including EGFR, ALK, and BRAF, as well as their potential mechanisms of drug resistance.