Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni(2+) affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins

Fusion of a Short Peptide that Binds Immunoglobulin G to a Recombinant Protein Substantially Increases Its Plasma Half-Life in Mice

<div><p>We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) <i>Science</i> 287∶1279–1283]. IgGBP fusion increases the <i>in vivo</i> half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the <i>in vitro</i> and <i>in vivo</i> properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in <i>Escherichia coli</i> and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn <i>in vitro</i>. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.</p></div

IgGBP fusion extends mKate half-life in hFcRn Tg mice when co-administered as a 1∶1 mol mixture with hIgG1 without altering hIgG1 clearance.

<p>(<b>a</b>) Schematic of the co-administration scheme. In this experiment, human FcRn Tg mice were not pre-dosed with exogenous hIgG1. Instead mKate-IgGBP and hIgG1 were pre-mixed in a 1∶1 mol ratio and co-injected via the tail vein. (<b>b</b>) Clearance of mKate-IgGBP in hFcRn Tg mice dosed alone (blue triangles) or co-dosed at a 1∶1 mol mixture with hIgG1 (yellow triangles). The % mKate-IgGBP remaining was calculated by normalizing the fluorescent emission at all time points to the maximum value observed in the first bleed 5 min after protein injection. (<b>c</b>) Clearance of labeled human IgG1 in hFcRn Tg mice dosed as a single agent via the tail vein (blue triangles) compared to the clearance of labeled hIgG1 co-administered as a 1∶1 mol mixture with mKate-IgGBP was measured to determine if bound mKate-IgGBP alters the eliminate profile of hIgG1 (red squares). The % hIgG1 remaining was calculated by normalizing the fluorescent emission at all time points to the maximum value observed in the first bleed 5 min after protein injection. Dashed lines in each panel represent the data fit to a 2-compartment PK model in Prism and the β-phase half-life shown in the figure was calculated as described in the Methods section. The data shown in each panel are the mean (n = 3 bleeds per time point) and error bars indicate s.d.</p

IgGBP fusion results in specific and high affinity binding to human IgG1 at a site overlapping the FcRn.

<p>(<b>a</b>) Competition ELISA between dIgG-HRP and unlabeled IgGs binding to mKate-IgGBP coated plates. (<b>b</b>) Sensograms demonstrating mKate-IgGBP (100 nM) binding to immobilized hIgG1 but not mIgG1 or rIgG2b. Unmodified mKate lacks binding to immobilized hIgG1 by SPR. (<b>c</b>) Competition of labeled hIgG1 accumulation in MDCK hFcRn-EYFP/hβ₂m cells at pH 6 by unlabeled hIgG1, mKate-IgGBP, and mKate. MDCK hFcRn-EYFP/hβ₂m cells were co-incubated with 1 µM labeled hIgG1-TAMRA and increasing concentrations of unlabeled hIgG1, mKate-IgGBP, and mKate for 1 hr at 37°C and analyzed by FACS as described in the methods section. The mean fluorescent intensity (MFI) of each test protein was normalized to the average MFI of hIgG1-TAMRA accumulation in MDCK hFcRn-EYFP/hβ₂m cells in the absence of unlabeled competitor and plotted as the % of hIgG1-TARMA accumulation as a function of competitor concentration. The data shown are the mean and error bars indicate s.d.</p

IgGBP fusion extends mKate half-life in hFcRn Tg hIgG+ mice.

<p>(<b>a</b>) Schematic of the hFcRn Tg hIgG1+ mouse model. hFcRn Tg mice were dosed i.p. with 500 mg/kg of recombinant hIgG1 48 hours prior to injection of mKate-IgGBP. (<b>b</b>) Clearance of mKate-IgGBP in wild-type (purple circles), hFcRn Tg (blue triangle), and hFcRn Tg hIgG1+ (red diamond) mice dosed i.v. at 10 mg/kg via the tail vein as a single agent. The % mKate-IgGBP remaining was calculated by normalizing the fluorescent emission at all time points to the maximum value observed in the first bleed 5 min after protein injection. Dashed lines represent the data fit to a 2-compartment PK model in Prism and the β-phase half-life shown in the figure was calculated as described in the Methods section. The data shown are the mean (n = 3 bleeds per time point) and error bars indicate s.d.</p

IgGBP fusion as a strategy to improve protein half-life by targeting serum IgG.

<p>(<b>a</b>) Schematic of genes encoding mKate or mKate modified at its C-terminus with an IgGBP sequence. * indicates thrombin cleavage site for removal of poly-histidine tag. (<b>b</b>) Cartoon depicting binding of mKate-IgGBP to the Fc-domain of hIgG and the corresponding crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102566#pone.0102566-DeLano1" target="_blank">[16]</a> of the IgGBP (green) in complex with Fc (red) (PDB 1DN2). Critical Fc amino acids that contribute to IgGBP binding at the C_H2–C_H3 interface are colored cyan. The same set of Fc residues is critical for FcRn binding. (<b>c</b>) Proposed half-life extension mechanism of IgGBP fusion. mKate-IgGBP binds serum IgG thus restricting its excretion through the kidney whereas unbound mKate-IgGBP is cleared through the glomerulus.</p