Synthesis and Characterization of Human Prorenin in Escherichia coli1

Abstract

DNA sequences encoding Ile-Glu-Gly-Arg and human prorenin were joined and placed under the transcriptional control of the Escherichia coli trp promoter-operator in the expression vector pTR501. E. coli cells transformed with pTR501 expressed high levels (30% of total cell protein) of prorenin as part of a hybrid protein with the trp E gene product. The chimeric protein, accumulated in a sedimentable form, was dissolved in 6 M guanidine X HCl, purified to near homogeneity, and renatured by dialysis. The complete prorenin sequence was then excised from the renatured hybrid protein using blood coagulation factor Xa, a proteinase which is highly specific for the tetrapeptide insert Ile-Glu-Gly-Arg introduced between the 9 amino-terminal residues of the trp E gene product and the first amino acid (Thr 1) of prorenin. Human prorenin thus obtained was readily activatable with trypsin and showed close similarities to naturally occurring prorenin in its biochemical and immunochemical properties.

Purification of recombinant human pepsinogens and their application as immunoassay standards

“…Other aspartic proteases [19,20,221 have been expressed as non-fused "inclusion bodies". Human PGs were not obtained as non-fused products in this study.…”

“…The individual expression and purification of recombinant PGA and PGC has, therefore, large advantages. Some aspartic proteases have been cloned in Escherichia coli and expressed as enzymatically active forms [19][20][21][22][23]. However, the application of recombinant PGs to the immunoassay system has not yet been reported.…”

Renin Inhibitor in Soybean

“…These human renins showed a heterogeneous electrophoretic pattern because of the variety of sugar chains and partial degradation. The expression of recombinant human prorenin in animal cells [Poorman et al, 1986, Weighous et al, 1986, Vlahos et al, 1990 or Escherichia coli cells [Imai et al, 1986] has also been reported. In the case of Chinese hamster ovary cells [Poorman et al, 1986], recombinant human prorenin was secreted into the medium.…”

“…However, the expression level was very low. On the other hand, with the expression of human renin in E. coli cells, the expressed human prorenin formed inclusion bodies and did not properly refold into active renin [Imai et al, 1986]. We constructed thioredoxin-human prorenin fusion protein expression vector.…”

Refolding and Activation of Human Prorenin Expressed inEscherichia coli: Application of Recombinant Human Renin for Inhibitor Screening

“…[13][14][15][16][17][18][19] On the other hand, with the expression of human renin in E. coli cells, the expressed human prorenin made inclusion bodies and did not properly refold into active renin. [20][21][22] The angiotensin I-converting enzyme (ACE) has been used as a target enzyme in RAS for screening inhibitors because of its simple assay method. However, renin is a rate-limiting enzyme in RAS, so it was not used because the measurement was so complicated.…”