Analysis of disuiphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disuiphide bridge in the mitogenic activity of the hormone

Abstract

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Evaluation of the Role of N-linked Oligosaccharides in Rat Placental Lactogen Action by Site-Directed Mutagenesis.

“…The assay is also useful for the analysis of structure-function relationships of lactogenic hormones [35]. The proliferative activities of lactogenic hormones for Nb2 cells are mediated by cell surface PRL-R [36].…”

Grass carp prolactin: Molecular cloning, tissue expression, intrapituitary autoregulation by prolactin and paracrine regulation by growth hormone and luteinizing hormone

“…In mammals, the "up-up-down-down" topography of helix H1-H4 in PRL is a typical feature of the members of GH/PRL family (Brooks, 2012). Besides, the disulfide bonds formed by conserved pairs of Cys residues not only play a role in maintaining the 3D protein structure but also contribute to the bioactivity of PRL, e.g., the mitogenic activity in NB2 cells (Luck et al, 1992) and osmoregulatory ability in urinary bladder (Doneen et al, 1979). Using in situ hybridization with riboprobe based on the PRL sequence obtained, PRL transcript expression was mapped to the RPD of the carp pituitary and overlapped with the distribution of lactotrophs with PRL immunoreactivity (Fig.…”

Hydrophobicity Engineering to Increase Solubility and Stability of a Recombinant Protein from Respiratory Syncytial Virus

“…Since it has been reported that cysteine to serine substitutions can lead to increased solubility of recombinant proteins (Luck et al, 1992), the two cysteine residues not included in the predicted essential loop formation were substituted for serine residues. A synthetic gene fragment encoding this peptide was assembled as described above and denoted GCys (Fig.…”

“…Rinas et al (1992) showed that a single cysteine residue substitution caused a dramatic decrease in solubility, while two other cysteine to serine substitutions did not alter the solubility properties. In contrast, Luck et al (1992) demonstrated that by replacing cysteine residues with serine residues in bovine prolactin, a significant increase in the protein solubility was achieved. Furthermore, Hellebust et al (1989) showed that the replacement of basic amino acids in a recombinant protein can dramatically enhance the proteolytic stability in vivo.…”

“…reported, despite the fact that dramatic differences in the structural andor functional properties of recombinant proteins can be obtained by single amino acid changes (Rinas et al, 1992;Cedergren et aI., 1993;Luck et al, 1992). In certain cases, specific modifications have been shown to alter the solubility andl or stability of the recombinant protein (Schein, 1993;Rinas et al, 1992;Luck et al, 1992;Hellebust et al, 1989;Strandberg and Enfors, 1991). Rinas et al (1992) showed that a single cysteine residue substitution caused a dramatic decrease in solubility, while two other cysteine to serine substitutions did not alter the solubility properties.…”