Methods for preparing human thrombopoietin polypeptides by mammalian cell cultures
View Patent ↗Procedures to produce a biologically active human TPO by mammalian cells in in vitro cultures are disclosed. Murine myeloma cells were genetically modified by introducing the gene of human thrombopoietin through a DNA construction which includes an immunoglobulin promoter associated to an immunoglobulin enhancer. The hTPO obtained is useful with methods of stimulating proliferation or development of hematopoietic cells of the megakaryocytic leneage in vitro and in vivo.
1. An expression vector replicable in mammalian cells, comprising the following elements grouped in a way that assures a correct and efficient transcription of a fragment comprising the complete sequence encoding the human thrombopoictin (hTPO) polypeptide including its signal peptide (eTPO):
(i) a hTPO transcription portion consisting of:
a) a transcription promoter of a variable kappa gene of human immunoglobulins
b) a DNA segment encoding the complete hTPO polypeptide including its signal peptide
c) an initnunoglobulin transcription enhancer DNA segment of the human kappa gene in a 3′ location with respect to hTPO gene, wherein said enhancer DNA segment consists of the DNA sequence 5′ to the Ck region of the human kappa gene
d) a transcription terminator
e) a polyadenylation signal; and
(ii) gene encoding resistance to the antibiotic neomicyn type drugs
(iii) a gene encoding resistance to the antibiotic ampicillin; and
(iv) a replication origin for E Coli (ColEI ori).
2. A cultured cell line derived from the munine myeloma called X-63 (ATCC: P63X63Ag8.6533, CRL 1580), which has been stably transfected by the expression vector described in claim 1 , and tat is capable of producing and secreting the mature recombinant human thrombopoietin (rhTPO) polypeptide towards the culture media in a biologically active form.
3. A method for obtaining a recombinant polypeptide corresponding to human thrombopoietin (hTPO), comprising the steps of:
(a) stably transfecting a cultured cell line derived from the murine myeloma called X-63 (ATCC; P63X63Ag8.6533, CRL 1580) with the expression vector described in claim 1 , and
(b) expressing the mature recombinant human thrornbopoietin polypeptide.