Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.
The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.
Libby
R T
RT
Braedt
G
G
Kronheim
S R
SR
March
C J
CJ
Urdal
D L
DL
Chiaverotti
T A
TA
Tushinski
R J
RJ
Mochizuki
D Y
DY
Hopp
T P
TP
Cosman
D
D
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005822:Genetic Vectors; D006098:Granulocytes; D006801:Humans; D008264:Macrophages; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003115:Colony-Stimulating Factors; D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA