Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.
A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.
Bautsch
W
W
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Emde
M
M
Kretzschmar
T
T
Köhl
J
J
Suckau
D
D
Bitter-Suermann
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015936:Complement C5a / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D015936:Complement C5a