Single-step purification of recombinant melanoma growth-stimulating activity by anion-exchange high-performance liquid chromatography.
The cytokine melanoma growth-stimulating activity (MGSA) is a growth factor for melanoma cells and a chemotaxin for neutrophils. Known purification procedures of MGSA from human sources or expression systems give a low yield and require multiple chromatography steps. Here, a fast and high-yield method for the purification of recombinant MGSA is described. Approximately 500 micrograms MGSA were recovered from the bacterial lysate of a 10 liter culture within a day. For this purpose, total mRNA of Hs294T melanoma cells was isolated and cDNA of MGSA was obtained by reverse transcription and polymerase chain reaction. The cDNA of MGSA was subcloned into the expression vector pGEX-2T, generating a fusion with the Schistosoma japonicum glutathione S-transferase gene. The fusion protein was expressed in E. coli DH5a and purified from the bacterial lysate using glutathione-sepharose beads. MGSA was cleaved from the complex of fusion protein and glutathione-sepharose beads with thrombin and purified to homogeneity by anion-exchange high-performance liquid chromatography with a Mono-S-column. The bioactivity of the recombinant MGSA was assessed by chemotactic migration and triggered [Ca2+]i-transients in human neutrophils. In addition, [125I]MGSA bound specifically to undifferentiated human leukemia cells HL-60 transfected with the cDNA of the interleukin-8 (IL-8) receptor beta with similar properties as [125I]IL-8. Thus, this described method might be a powerful tool to generate large amounts of cytokines in a short time.
Norgauer
J
J
Department of Dermatology, Albert-Ludwigs-Universität, Freiburg, Germany.
Metzner
B
B
Parlow
F
F
Elsner
J
J
Kownatzki
E
E
Schraufstätter
I U
IU
D000595:Amino Acid Sequence; D001483:Base Sequence; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D005784:Gene Amplification; D006133:Growth Substances / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007951:Leukemia, Myeloid / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D004273:DNA, Neoplasm; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins