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.

Expression of Trichoderma reesei and Trichoderma viride xylanases in Escherichia coli.

Synthetic genes encoding the 190 amino acid Trichoderma reesei xylanase II (TrX) and the closely related Trichoderma viride xylanases have been synthesized in a two-step procedure. Initially, a partial gene encoding amino acids 92-190 was constructed in fusion with the N-terminal half of the Bacillus circulans xylanase (BcX). The remaining BcX gene sequence was replaced during the assembly of the coding sequence for amino acids 1-91.

Effect of base-mismatch in the cohesive ends of oligonucleotide in gene cloning.

The tolerance of mismatched nucleotides between the cohesive ends of insert and target DNAs in gene cloning has been investigated. An oligonucleotide duplex with a cohesive end GGCC-5' or variation was ligated to the 5'-CCGG end of a linearized plasmid. The ligation mixture was used in the transformation of E.

Overexpression of the Bacillus subtilis and circulans xylanases in Escherichia coli.

An efficient expression system for a low-molecular mass xylanase in Escherichia coli has been developed. A gene encoding the mature Bacillus circulans (Bc) xylanase was designed to imitate the frequency of degenerate codons used in E. coli.

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.