Christine E. Smith

Characterization and expression of an antifungal zeamatin-like protein (Zlp) gene from Zea mays.

A cDNA clone encoding a basic thaumatin-like protein of Zea mays was recovered from a mid-development seed cDNA library. The gene, Zlp, encoded a protein that was nearly identical with maize zeamatin and [alpha]-amylase/trypsin inhibitor. Expression of Zlp mRNA was highest in the endosperm tissue of seed 4 weeks after pollination. Expression of zeamatin-like (ZLP) protein correlated with mRNA; also, a low basal level of ZLP expression in leaf was not appreciably induced by abiotic stresses. ZLP was expressed with its own signal peptide in insect cells and in transgenic Arabidopsis and tomato plants. ZLP was secreted in all three systems, with correct processing of the signal peptide. ZLP expressed in transgenic tomato was found to be partially subjected to a proteolytic cleavage after residue 180, by an unknown mechanism, to give a “nicked” isoform of ZLP. Purified ZLP from all three sources, as well as purified “nicked” ZLP from tomato, demonstrated fungal inhibition against Candida albicans and Trichoderma reesei, with marginal inhibition observed against Alternaria solani and Neurospora crassa.

Mistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons.

Partial misincorporation of Lys for Arg has been observed for the Arg residues of IGF-1 when the molecule is expressed in Escherichia coli using a synthetic gene with the low frequency AGA codon encoding all six Arg residues and yeast preferred codons encoding the remaining residues. The Lys for Arg substitution at these residues could not be detected when a gene containing E. coli preferred codons, with the codon CGT coding for all Arg residues, was used for the expression of the protein. Similarly, no misincorporation of Lys for Arg could be detected when a gene containing Escherichia coli preferred codons at all positions, except for an AGA codon at Arg (36), was utilized.

In vitro and in vivo activity of chymotrypsin-activated big endothelin (porcine 1–40)

We investigated whether big endothelin (porcine 1-40) had contractile activity in isolated rat aorta or pressor activity when injected intravenously into the anesthetized rat. When isolated rat aorta was exposed to a 100 nM concentration of big endothelin, 4.8% of a maximal KCl contraction was observed, compared to 131% of KClmax when paired aortic rings were exposed to an equivalent concentration of synthetic endothelin. Likewise, big endothelin had very weak pressor activity when injected intravenously into anesthetized, ganglion-blocked rats at 10 nmol/kg. When big endothelin was incubated with chymotrypsin, native endothelin and other peptide fragments were formed. Chymotrypsin-treated big endothelin produced an endothelin-like contraction when applied to isolated rat aortic rings, and a characteristic endothelin-like effect on blood pressure in vivo. Our results indicate that the biological activity of endothelin could be effectively blocked by inhibiting the enzyme which converts big endothelin to endothelin.

Isolation and characterization of a 25 kDa antifungal protein from flax seeds.

We have purified a 25 kDa protein from flax seeds to homogeneity by polyethyleneimine precipitation, ammonium sulfate precipitation, chitin extraction, Mono S cation exchange and C18 reversed phase column chromatographies. The purified protein strongly inhibited the growth of the agronomically important pathogen Alternaria solani, the causative agent of tomato early blight and in synergy with nikkomycin Z strongly inhibited the human pathogen Candida albicans. Amino terminal sequence analysis of the purified protein indicated that it has a high degree of homology to other reported pathogenesis-related antifungal proteins.

Carboxy-terminal Protein Sequence Analysis using Carboxypeptidase P and Electrospray Mass Spectrometry

Publisher Summary This chapter discusses carboxy-terminal protein sequence analysis using carboxypeptidase P (CPP) and electrospray mass spectrometry (ESMS). To test the applicability of the ESMS analysis method for the CPP digestion of proteins, recombinant E. coli derived human IL-3 was chosen as a model. IL-3 has a C-terminal sequence of …T-T-L-S-L-A-I-F-OH. The digestion conditions were used to digest 2.7 nmoles of IL-3. Aliquots of 500 pmoles were removed at several time points—10 min, 20 min, and 50 min—and analyzed by ESMS. At the 10 min time point, the appearance of multiply-charged ions representing consecutive losses of five C-terminal amino acids was observed in the mass spectrum. The average molecular weight of each truncated species can be determined through deconvolution of the mass spectrum. The chapter shows a closeup view of the (M+9H)+9 charge state of CPP-digested IL-3. The mass differences of these ions reflect the sequential loss of amino acids from full-length IL-3.

Isolation of Escherichia coli synthesized recombinant proteins that contain ε-N-acetyllysine

Publisher Summary This chapter describes the presence of significant amounts of ɛ-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. ESMS, amino acid sequencing and amino acid analyses demonstrate the presence of ɛ-N-acetyllysine in two recombinant proteins; bovine placental lactogen and human tissue factor pathway inhibitor. These data establish that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E.coli . Formation of ɛ-N-acetyllysine in eukaryotic systems involves a post-translational mechanism in which the acetyltransferase uses acetyl-CoA as the source of the acetyl group. It may be possible that acetylation of lysines occurs by a chemical mechanism with acetyl-CoA or some other metabolic intermediate providing the source of the acetyl group. The chapter explains that acetylation of lysines is an important modification, which can occur during the expression of recombinant proteins expressed in E.coli. However, investigations into the effects of fermentation conditions on the level of ɛ-N-acetyllysine formation may lead to a better understanding of this event.

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.