Richard J. Kirschner

Gene synthesis, E. coli expression and purification of the bovine growth hormone releasing factor analog, (Leu27,Hse45) bGRF

Abstract The DNA sequence coding for an analog of bovine growth hormone releasing factor (bGRF), having the methionine at position 27 substituted by leucine, was chemically synthesized with flanking methionine codons. Tandem ligations of this sequence allowed the monomer or fusion proteins of 2, 4, or 8 bGRF units to be expressed in E. coli from a pBR322-based vector. Although the monomer and dimer genes do not generate detectable products by SDS-PAGE, the fusion products of 4 and 8 bGRF accumulate as insoluble aggregates representing over 20% of the cellular protein; there appears to be decreased protease susceptibility as the fusion protein increases in size. The inclusion body material was solubilized in formic acid and octameric bGRF was treated with cyanogen bromide to release the monomer containing the natural amino-terminal tyrosine and an unnatural carboxy-terminal homoserine lactone. The reaction conditions represented a compromise between minimizing a formic acid-dependent modification of bGRF and achieving complete digestion. About 150 g of the bGRF analog were isolated at ≥99% purity using ion exchange and preparative reverse-phase chromatography. Intravenous injection of this peptide into steers stimulates somatotropin release as measured by significant increases in serum concentrations of the hormone.

The expression, affinity purification and characterization of recombinant pseudomonas exotoxin 40 (pe40) secreted from escherichia coli

Procedures have been devised for producing high yields of purified recombinant PE40, a protein important for the development of the anti-AIDS therapeutic, sCD4-PE40. PE40 is a truncated form of the bacterial toxin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but retains domains II and III that are involved in membrane translocation and inhibition of protein synthesis in eukaryotic cells. Expression vectors in Escherichia coli encoding PE40 synthesized the product as a soluble protein under control of the T7 promoter. The expression capabilities of transformants of E. coli BL21(DE3) were highly unstable. Expression levels (secreted and total) were evaluated in shake flasks and at the 10-1 scale at 27 degrees C and 37 degrees C, and following induction by IPTG or lactose. The cell-free media from the batch process was applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity. This purification strategy was based on the structural similarity of the blue dye to NAD, a natural substrate for domain III of PE40. Green and red dye-ligand chromatography steps removed nicotinamide as well as minor residual E. coli proteins from PE40. Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ionization mass spectrometry to determine the sequence and verify disulfide bonding.