Abstract
Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.
Highlights
Specific degenerate codons in the amino-terminal postulated that the low yield was due to instability of both region of a synthetic human parathyroid hormone PTH and its mRNA(Morelle et al, 1988)
With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH productat 6.5 mg/ liter
Composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH
Summary
Regions of the synthetic gene were reconstructed to imitate the E. coli-favored codon degeneracy. Plasmid pPTH-AA(Table l),might haveweakened such the prepro-PTH sequence was unable to facilitate secondary structure (AG = -3.2 kcal) and imsecretion andprocessing of P T H in yeast (Bornet al.; 1987a) proved P T H expression Such mechanism is inadeand Escherichia coli (Born et al, 1987b). Direct expression of the PTH cDNA (minus the production by the plasmids pPTH-AA and pPTH-AA-Eco prepro sequence) intracellularly generated immunoreactiove (Tables 1 and 2) contradictsearlier conclusions that PTHat 0.2 mg/literin E. coli (Breyel et al, 1984). It was tandem repeats of rare degeneratecodons (Varenne andLazdunski, 1986) andtheir proximity totheinitiation codon. Konishi of Biotechnology Research Institute, for obtaining mass spectra, and Jennifer Pendlebury of Allelix Biopharmaceutics (Mississauga, Canada) for conducting the bioassay studies
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