Immunobiol., vol. 185, pp. 41-52 (1992)
1 Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover, and 2 Fakultat fur Chemie, Universitat Konstanz, Konstanz, Germany
Human C5a Anaphylatoxin: Gene Cloning and Expression in Escherichia coli WILFRIED BAUTSCH 1, MONICA EMDE 1 , TITUS KRETZSCHMAR!, JORG KOHL 1, DETLEV SUCKAU2 , and DIETER BITTER-SUERMANN! Received August IS, 1991 . Accepted in Revised Form February S, 1992
Abstract A gene coding for the human anaphylatoxin CSa 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 pKK233-2 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 ~ mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with CSa-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 CSa per liter bacterial culture.
Introduction The complement system consists of a set of plasma proteins and their corresponding cellular receptors which act as a major immunological defense barrier against foreign substances. Activation of the complement system by e.g. antigen-antibody complexes or bacterial surfaces triggers an amplification cascade of proteolytic cleavage and protein assembly events of the complement components which ultimately leads to the destruction and final elimination of the foreign body (1). CSa, a small glycoprotein of 74 amino acids, is cleaved from the a-chain of the complement protein CS during activation of the complement system (2). Though not primarily involved in antigen inactivation and metabolism it mediates a variety of inflammatory (
1 Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover, and 2 Fakultat fur Chemie, Universitat Konstanz, Konstanz, Germany
Human C5a Anaphylatoxin: Gene Cloning and Expression in Escherichia coli WILFRIED BAUTSCH 1, MONICA EMDE 1 , TITUS KRETZSCHMAR!, JORG KOHL 1, DETLEV SUCKAU2 , and DIETER BITTER-SUERMANN! Received August IS, 1991 . Accepted in Revised Form February S, 1992
Abstract A gene coding for the human anaphylatoxin CSa 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 pKK233-2 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 ~ mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with CSa-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 CSa per liter bacterial culture.
Introduction The complement system consists of a set of plasma proteins and their corresponding cellular receptors which act as a major immunological defense barrier against foreign substances. Activation of the complement system by e.g. antigen-antibody complexes or bacterial surfaces triggers an amplification cascade of proteolytic cleavage and protein assembly events of the complement components which ultimately leads to the destruction and final elimination of the foreign body (1). CSa, a small glycoprotein of 74 amino acids, is cleaved from the a-chain of the complement protein CS during activation of the complement system (2). Though not primarily involved in antigen inactivation and metabolism it mediates a variety of inflammatory (
