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

Abstract

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.

Figure 1. Schematic depicting the construction of pET22b-pelB hGH (left) and pET22b-ompA hGH (right) expression vectors

The gene encoding hGH was PCR amplified as described in Materials and Methods and restriction cloned into the pET22b E. coli expression vector.

Figure 2. Reducing and non-reducing SDS-PAGE analysis of recombinant hGH

Reduced TEV-TROPIN (lane 1), non-reduced TEV-TROPIN (lane 2), reduced pelB-hGH (lane 3), non-reduced pelB-hGH (lane 4), reduced ompA-hGH (lane 5), non-reduced ompA-hGH (lane 6). 7.5 μg of protein were loaded in each lane.

Figure 3. Size exclusion chromatography (a–c) and MALF-TOF (d–f) analysis of TEV-TROPIN (a, d), pelB-hGH (b, e), and ompA-hGH (c, f)

The retention time of the SEC calibration standards are indicated by tick marks on the x-axis. The standards include thyroglobulin 670 kDa, gamma-globulin 158 kDa, ovalbumin 44kDa, myoglobin 17 kDa, and vitamin B12 1.35 kDa. The unlabeled tick under the hGH peak in each panel corresponds to the retention time of the 17 kDa myoglobin standard. Obs: = Observed, Exp: = Expected.

Figure 4. In vitro hGH receptor binding ELISA (a) and Nb2 cell potency bioassay (b)

Each data point represent the mean (n=3) and error bars indicate standard deviation (s.d.). The dashed line indicates data fit to a log(agonist) vs. response model using Prism for derivation of K_D and EC₅₀. Data points were normalized to the maximum observed hGH receptor binding (a) or Nb2 cell count (b) and plotted as percentage of maximum binding or growth. The data shown in (a) and (b) are representative of at least 3 independent protein preparations.

Figure 5. Surface plasmon resonance sensograms of hGH binding to immobilized hGH-R

Increasing concentrations of TEV-TROPIN, pelB-hGH, or ompA-hGH (0.78 nM to 50 nM) were injected over immobilized hGHR-Fc at pH 7.4 (left) or pH 6 (right) as described in Materials and Methods. The resulting sensograms were fit to a steady state affinity model for derivation of K_D. All data were baseline-adjusted and reference cell-subtracted.