Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector

Abstract

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX 1-' day-' into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows two differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX.The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1 .Hemophilia B, an X-linked bleeding disorder, is due to a deficiency in blood-clotting factor IX. Factor IX is synthesised as a preprozymogen in the liver and is subjected to four post-translational modifications : glycosylation, NH2-terminal processing, vitamin-K-dependant 4-carboxylation of the first 12 glutamic acid residues and 3-hydroxylation of an aspartic acid residue [l -31. Due to these complexities, factor IX production using recombinant DNA technology requires the use of mammalian cells as expression host. Several mammalian cell lines have been tested for their ability to produce active human factor IX: baby hamster kidney cells [4], rat hepatoma cells [5], human hepatoma cells [6], CHO cells deficient in dihydrofolate reductase [7]. More recently, using a different approach, the complete factor IX cDNA was expressed in transgenic mice [8]. The isolation and characterisation of the recombinant molecule have been previously described in only one study [7].In this paper we report the expression of human recombinant factor IX in a stable wild-type CHO cell line transformed with a dicistronic expression vector. The recombinant molecule has been purified, N-terminally sequenced, the ex- tent of 4-carboxylation of glutamic acid determined, and carbohydrate composition compared to the natural molecule. MATERIALS AND METHODS Construction of factor I X expression vector, pTG381Factor IX cDNA was recovered fr...

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

“…fIX secreted from transfected CHO cells is partially y-carboxylated and the propeptide is incompletely cleaved (13). Even in cell types such as BHK21, capable of synthesizing other vitamin K-dependent 'y-carboxylated proteins, the fIX produced exhibited only partial activity (14), although human 293 cells have been reported to secrete active fIX at relatively modest levels (26).…”

“…Its normal site of synthesis is the liver where it undergoes several posttranslational modifications required for activity including propeptide cleavage, 13-hydroxylation, and y-carboxylation. The production of recombinant fIX in cell culture has proved problematical in the past because many mammalian cell lines are unable to carry out these modifications effectively (12)(13)(14). This laboratory has described transgenic sheep (15) and transgenic mice (10) DNA Constructs.…”

Protein Design and the Development of New Therapeutics and Vaccines

“…In order to more fully characterize FVIIILlII, recombinant cell lines expressing either complete FVIII or FVIIILlII were established by transfecting CHO cells with an expression vector capable of expressing heterologous genes in mammalian cells (Balland et at., 1988), into which the appropriate cDNA was integrated. The (I I I I I)· Cleavage sites are shown for thrombin (Ha) and the unknown protease (X) that cleaves between amino acids 1648-1649 (the latter deleted in FVIII8H).…”

Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies

“…This selectable marker was included in a single expression vector along with the antibody heavy and light chain genes driven by the human CMV promoter (Boshart et al, 1985). Use of the gpt gene as a selectable marker in numerous cell types has been described previously (Balland et al, 1988;Lupker et al, 1983;Ringold et al, 1981;Wang et al, 1989). GPT catalyzes purine biosynthesis via the salvage pathway, by converting xanthine to xanthine monophosphate, in medium containing xanthine and MPA to inhibit endogenous inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway.…”