The 9E10 antibody epitope (EQKLISEEDL) derives from a protein sequence in the human proto-oncogen p62(c-myc) and is widely used as a protein fusion tag. This myc-tag is a powerful tool in protein localization, immunochemistry, ELISA or protein purification. Here, we characterize the myc-tag epitope by substitutional analysis and length variation using peptide spot synthesis on cellulose. The key amino acids of this interaction are the core residues LISE. The shortest peptide with a strong binding signal is KLISEEDL. Dissociation constants of selected peptide variants to the antibody 9E10 were determined. scFv constructs with the shortest possible myc-tags were successfully detected by Western blot and ELISA, giving a signal comparable to that of the original myc-tag.

A new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a step-wise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.

No abstract

Reverse transcriptase from feline immunodeficiency virus (FIV) has been cloned and expressed in Escherichia coli. We have purified this recombinant enzyme and shown that it is a 66-kDa protein that is indistinguishable from virion-derived FIV reverse transcriptase in sensitivity to the 5'-triphosphates of 3'-azido-3'-deoxythymidine and the four 2',3'-dideoxynucleosides. The availability of large quantities of the FIV reverse transcriptase will allow more detailed physical and pharmacological studies.

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic a-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacilus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known oa-amylase sequences. We found 83% homology with the 48-kDa a-amylase part of the Bacilus polymyxa Pj-a-amylase polyprotein and 50%o homology with Taka amylase A of AspergiUlus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacilus amyloliquefaciens a-amylase gene. A number of thermostable enzymes have been isolated from Thermoactinomyces vulganis strains. Among these were thermitase (4, 13, 14, 28, 29), a protease from the subtilisin family (2, 5, 36), and a variety of oa-amylases (pullulanases). The a-amylases of strains R47 and 42, like fungal glucoamylases (1, 15, 49, 54), hydrolyze starch and pullulan (1, 54). The a-amylase of T. vulgaris 94-2A, however, utilizes only starch and glycogen as substrates, not pullulan. a-Amylase 1 of T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa and was previously shown to exhibit striking homology to Taka amylase A of Aspergillus oryzae for a short N-terminal sequence (22, 60, 67). Smaller peptides of 33 and 18 kDa have been shown to be products of limited AmyTV1 proteolysis (21). The AmyTV1 amylase is unusual because of its temperature optimum at 62.5°C in a low pH range (4.8 to 6), its relatively short half-life of about 5 min at 70°C, and the production of maltose and maltotriose in the hydrolysate, which lacks glucose (47). In order to overcome problems of poor growth and product yield in T. vulgaris 94-2A, we cloned the gene encoding the AmyTV1 amylase and performed expression studies with Bacillus subtilis. The transcription start site(s) of the gene in B. subtilis was studied after deletion of the 5'-flanking region and after its replacement with the promoter of the B. amyloliquefaciens cx-amylase gene. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The T. vulgaris strain used was originally isolated and described by Klingenberg et al. (29, 47). The strain 94-2A was selected for higher enzyme production (29). The EMBL3 lambda phage (12) was used for preparation of a T. vulgaris DNA bank in Escherichia