Biologically active bone morphogenetic protein-2 (BMP-2) has been produced from Escherichia coli through in vitro refolding of inclusion bodies, but the refolding procedure is complicated and the overall yield is low or the refolding buffer contains additional reagents. Our goal was to develop a new, simple and inexpensive method to improve the production of biologically active hBMP-2. The design of a 345-bp synthetic hBMP-2 gene was based on the amino-acid sequence of hBMP-2. A DNA sequence containing 11 restriction sites located approximately every 30 bp throughout the entire length of the coding region was selected from the large number of possibilities. The codon usage of the resulting BMP-2 gene was modified to include those triplets that are utilized in highly expressed E. coli genes. A ribosome binding site was added 10 bases upstream of the coding region to direct the initiation of translation in E. coli. The spacer region (-1 to -9) was made A/T rich to reduce potential mRNA secondary structure in the vicinity of the translation start site. The hBMP-2 gene was assembled into pUC18, subcloned into pCold I vector, and over-expressed as a soluble protein in Origami B(DE3). hBMP-2 was homogeneously purified using heparin-Sepharose and FPLC Resource-Q column. hBMP-2 dimer (24 kDa) possessed similar level of activity as hBMP-2 from CHO cells, whereas hBMP-2 monomer (12 kDa) did not show any biological activity.