Anthony J. Salerno

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

Cloning, expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a relative molecular mass of 25 000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein.

Tissue distribution and excretion of 2,4‐[14C] toluenediamine in the mouse

The tissue distribution and excretion of 2,4-[14C]toluenediamine was studied in male mice given a single ip dose (1 microCi, 0.667 mg/kg). By 24 h 52% of the administered radioactivity had been excreted in the urine and 22% in the feces. The organs with the highest concentrations of radioactivity were the liver and kidneys. High concentrations of radioactivity were also observed in the gastrointestinal tract. Elimination of radioactivity from the liver, kidneys, and blood was biphasic, with half-lives of 11.7, 9.1, and 12.6 h, respectively, for the slow phases. The dominant route of excretion was via the kidneys; during the first hour after dosing, nearly 50% of the administered radioactivity was recovered in the urine. However, only an additional 2-4% of the dose appeared in the urine during the remaining 23 h of the experiment. By 24 h, only 1.25% of the administered radioactivity has been trapped from the air expired by the animals.

Synthetic Mussel Adhesive Proteins

The development of a technology for the biological and chemical synthesis of analogs to the adhesive protein of the mussel Mytilus edulis is described. This protein consists mainly of the repeating decapeptide sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys.

Expression of a Synthetic Mussel Adhesive Protein in Escherichia Coli

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a MW of 25,000 was expressed from one 600-bp gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. In strains employing T7 RNA polymerase for induction, BP was produced at levels approaching 60% of total cell protein. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein as shown by amino acid composition and N-terminal sequencing to give an authentic consensus precursor protein. Although the repetitious gene containing 30-bp repeat units appeared stable in T7-based host/vector systems, it was less stable in a λPL promoter-based host/vector system. Codon diversification was examined as a potential method to alleviate the problems by constructing a repetitious gene comprised of 120-bp repeats. This longer repeat unit failed to confer additional stability upon the repetitious gene.