Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli.

Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate.

Gene fragment polymerization for increased yield of recombinant HIV fusion inhibitor enfuvirtide. [corrected].

Fuzeon (Enfuvirtide, T20) is the first fusion inhibitor approved by the FDA of the USA for the treatment of HIV/AIDS in combination with other anti-retroviral drugs. Enfuvirtide is a synthetic peptide that blocks the entry of HIV into healthy host CD4 cells, which requires very high (90 mg twice daily) therapeutic doses. To increase the yield of Enfuvirtide, a gene polymerization strategy was introduced and recombinant T20 (rT20) was expressed in Escherichia coli as a five copy repeat polypeptide with a histidine-tag. The five copy rT20 was purified by Ni-affinity chromatography and cleaved to single rT20 units by cyanogen bromide. Finally, single rT20 units were purified by reversed phase chromatography giving a yield (400 mg/l) with a purity >95 %, which exhibited specific biological activity similar to Fuzeon.

Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli.

The use of antiretroviral drugs is gaining importance in the recent past for the treatment of human immunodeficiency virus infection. Enfuvirtide (T20) is one of the fusion inhibitors, inhibits the fusion between the virus and healthy target CD4 cells. The treatment with T20 involves very high therapeutic dose. In addition to its high dose, production of T20 by synthetic methods is expensive and cumbersome. We report an alternative recombinant approach for the production of the T20 peptide through a novel short fusion-tag expression system. This expression system consists of the hydrophobic region of growth hormone (GH) as the fusion tag, a factor Xa cleavage site upstream to the T20. The fusion protein was expressed in Escherichia coli as inclusion bodies. We also report here, a simple and an efficient down-stream strategy for the purification of recombinant T20 peptide (rT20). Our study is the first to demonstrate a novel approach using GH fusion tag, ensured the peptide stability, for the production of rT20 which yields more than 250mg/L with 98% purity. The biological activity of the rT20 is comparable to its synthetic counterpart. Thus, this novel approach could be an alternate method of choice for production of therapeutically important small peptides.