Overexpression and characterization of recombinant human fusion protein IL-6/IL-2 (CH925).
An expression vector encoding the human recombinant fusion protein interleukin 6/interleukin 2 (IL-6/IL-2) was constructed. When a flexible linker had been synthesized and ligated with the IL-2 gene fragment by polymerase chain reaction (PCR) amplification, the IL-6 gene fragment was unidirectionally inserted into the upstream of the linker-IL-2 sequence. The molecule of the IL-6-linker-IL-2 fusion gene named E. coli DH5 alpha/pfIL-6/2 was cloned and identified by DNA sequencing. The expressed protein named as CH925 showed a strong band on SDS-PAGE and amounted to 32% of total cell protein, and its estimated molecular weight was about 37 kDa. The fusion protein purified by gel filtration and reversed-phase HPLC showed as almost homogeneous. CH925 possesses both IL-2 and IL-6 activities when assayed by CTLL2- and 7TD1-dependent cell lines, respectively. The specific activity of IL-2 was 2.1 x 10(6) U/mg while that of IL-6 was 2.3 x 10(8) U/mg. Our studies exhibited that CH925 exerted a significant augmentative effect on the growth of erythroid colony forming units (CFU-E), and synergized with erythropoietin (EPO) and/or IL-3 in a dose-dependent way. Our experimental results also showed CH925 at a low dose causing active lymphokine-activated killer (LAK) cell proliferation more vigorous than IL-2 and/or IL-6 (p < 0.001). CH925 is a novel fusion protein, being neither IL-6 nor IL-2, more potent than IL-2 and/or IL-6 and causing non-IL-2 and non-IL-6 functions of strong EPO-like and mild IL-3-like effects on erythroid progenitor cell growth. There is a potential for efficacious clinical application of CH925.
Zhao
C
C
Department of Experimental Hematology, Institute of Basic Medical Sciences, Beijing, China.
Tang
P
P
Wang
J
J
Mao
N
N
Jiang
F
F
Li
X
X
Liu
X
X
Zhang
M
M
Ren
Y
Y
Du
D
D
D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D017931:DNA Primers; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015979:Killer Cells, Lymphokine-Activated / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D017931:DNA Primers; D007376:Interleukin-2; D007377:Interleukin-3; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; C089276:recombinant fusion protein IL-6 - IL-2; D004921:Erythropoietin