Periplasmic Expression of a Novel Human Bone Morphogenetic Protein-7 Mutant in Escherichia coli

Abstract

Background: Bone Morphogenetic Proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 (BMP-7) was engineered through substitution of the BMP-7 N-terminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans (HSPGs).

Methods: The engineered protein was expressed in Escherichia coli (E.coli). The PelB signal sequence was used to translocate soluble proteins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein.

Results: The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml.

Conclusion: The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs.

Keywords: Bone morphogenetic protein-7 (BMP-7); Escherichia coli; Periplasmic expression.

Figure 1

Schematic representation of designed cassette gene. His -Tag coding sequence was engineered to the C-terminal of the construct in order to facilitate purification using Ni-NTA technology

Figure 2

A) The map of recombinant plasmid (pET_ B2BMP7). The size of expression construct was 5915 bp, B) Restriction analysis of recombinant plasmid. Lane 1: Size marker; lanes 2, 3: Digestion of pET_B2BMP7 with NotI or EcoRV created linear plasmids (5915 kb); lane 4: Digestion of recombinant plasmid with NotI/EcoRV created two fragments with expected sizes at 4095 bp and 1820 bp

Figure 3

A) SDS-PAGE analysis of a recombinant clone producing novel mutant. Lane 1, Protein marker SM0671; lanes 2, 3, two and four hours samples (post induction); lane 4, pre-induction sample. Arrows indicate the bands related to the pre-protein (pelB-hB2BMP-7). B) Western blot analysis of recombinant protein. Lane 1, pre-induction sample; lanes 2, 4, two and four hours samples (post induction); lane 3, Protein marker

Figure 4

A) SDS-PAGE analysis of the periplasmic extract and cell-pellet fraction. Unprocessed pre-protein (∼18 kDa) and processedmature protein (∼16 kDa) are indicated as (p) and (m) respectively. Lane 1, Protein marker SM0671; lane 2, Cell-pellet fraction; lane 3, Periplasmic extract, B) SDS-PAGE analysis of the purified protein. Lane 1, Concentrated purified mutant; lane 2, Protein marker, C) Western blot analysis of the mutant, Lane 1: purified mutant; lane 2: Protein marker; lane 3:pre-induction sample as negative control

Figure 5

Alkaline phosphatase release assay. The purified monomer did not show any significant biological activity similar to the commercial BMP-7-His. As positive control, commercially available rhBMP-7 produced in CHO cells was used. Each value is the mean of triplicates shown±SD

Figure 6

Heparin binding assay of BMP-7-His and B2BMP-7. The results showed dose-dependent binding of commercial BMP-7-His and its mutant to heparin. The background binding related to the non-heparin coated wells as negative control was considerably lower. Each absorbance value is the mean of triplicates shown±SD