Diana M. Zahab

High-pressure infrared spectroscopic study of human proinsulin gene expression in live escherichia coli cells

Infrared spectra of E. coli strain JM103 and transformants which overproduced recombinant proinsulin have been measured as a function of pressure up to 38 kbar. It is the first time that high-pressure infrared spectra of live bacteria have been successfully measured. In ambient conditions, spectra of the host strain JM103 and the transformants are generally identical. However, under pressure, distinct shifting pattern can be observed in specific spectral parameters of transformants, presumably due to accumulation of proinsulin in form of cytoplasmic inclusion bodies. In particular, the pressure-induced frequency shift of the amide III band (1235 cm-1) in the proinsulin-producing transformants is much smaller than in the host JM103. This pressure effect can potentially be an efficient approach to monitor maximum gene expression in microorganisms. Contrary to predictions based on model system, the pressure-induced denaturation and the sharp transition from disordered liquid crystalline state to the ordered gel state commonly observed in the aqueous solution of protein and aqueous bilayer dispersion of lipids, respectively, do not occur in the bacterial proteins and cell membrane of E. coli.

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1‐34)‐Asp‐Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72‐amino acid) fusion product of hPTH(1‐34)‐Asp35‐Pro36‐X, where X is a 36‐residue “arbitrary” downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH‐(1‐35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half‐maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH‐(1‐34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH‐(1‐35) was fully active as a bone anabolic agent.

Internal ribosome-binding site directs expression of parathyroid hormone analogue (8-84) in Escherichia coli.

Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.