Expression of a Cloned Human Fibrinogen cDNA inEscherichia coli: Synthesis of an A Alpha Polypeptide

Abstract

The construction of a plasmid, p166.9, for the controlled synthesis of the A alpha-chain of human fibrinogen in Escherichia coli is described. The plasmid combines the tac promoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from an E. coli plasmid beta-lactamase gene and a cDNA of the A alpha-chain of human fibrinogen. The tac promoter is repressed in lacIQ strains of E. coli and induced by isopropylthio-beta-D-galactoside (IPTG). Protein blot analysis of lysates of cells carrying p166.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A alpha-chain of human fibrinogen. The largest and predominant species corresponds to an apparent molecular weight of 63,000. When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A alpha-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA. Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A alpha, this indicates that the A alpha-peptide concentration in the culture media is 13 micrograms/ml.

The purification of eukaryotic polypeptides synthesized in Escherichia coli

“…While secretion of proteins from E. coli obviates the insolubility phenomenon, until recently expressed yields have been low. The production of milligram amounts per litre of ,-endorphin (Nagahari et al, 1985) and A a-fibrinogen (Lord, 1985) show that E. coli has potential as a secretion system. For further development this secretion mechanism must be elucidated.…”

“…Another eukaryotic polypeptide secreted at significant levels into the medium by E. coli was A a-fibrinogen (Lord, 1985). This polypeptide has a predicted Mr of 67000.…”

Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae

“…DNA of each chain was amplified from cDNA encoding the Aα-, Bβ-, and γ-chains of human fibrinogen and cloned into pMAL-C2X (New England Laboratory) as described previously (29–31). The recombinant proteins were purified by affinity chromatography with amylose resin, according to the manufacturer's instructions (New England Biolabs).…”

Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

“…cDNAs encoding the Aα-, Bβ- and γ-chains of human fibrinogen were generously provided by Professor Susan Lord (University of North Carolina at Chapel Hill) [38]–[40]. The full length and truncated forms of chains were amplified and cloned into pMAL-C2X (New England Laboratory) to express MalE-tagged versions of the chains.…”