A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

https://www.pnas.org/content/pnas/85/16/5879.full.pdf

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation.

Nutritional properties of microalgae for mariculture

Defensive genes in plants can be activated by several different types of nonpeptide signaling molecules. An endogenous polypeptide, consisting of 18 amino acids, was isolated from tomato leaves and was able at very low concentrations to induce the synthesis of two wound-inducible proteinase inhibitor proteins when supplied to young tomato plants. The sequence of the polypeptide was determined, and an identical polypeptide was synthesized that possessed full inducing activity. These data establish that a polypeptide factor can initiate signal transduction to regulate the synthesis of defensive proteins in plant tissues.

https://science.sciencemag.org/content/sci/253/5022/895.full.pdf

A Polypeptide from Tomato Leaves Induces Wound-Inducible Proteinase Inhibitor Proteins

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells

Rapid analysis of amino acids using pre-column derivatization.

Refined methods for separating PTC-amino acids on reverse phase columns may pose a challenge to traditional ion exchange techniques.

PITC derivatives in amino acid analysis

A new approach to the analysis of free amino acids and amino acids from hydrolyzed foods is described. The method is based on reaction of the free amino acids with phenylisothiocyanate to form stable derivatives which are subsequently separated by liquid chromatography. Sample preparation procedures are described and results are compared with conventional ion exchange results. Reproducibility of the new method has been determined on a typical food type sample.

A new, rapid, high-sensitivity analysis of amino acids in food type samples.

Understanding the mechanisms by which diseases occur is a complex process, and often involves the isolation and characterization of minute quantities of rare proteins from living tissue. Amino acid analysis is a vital tool for the elucidation of the structure of such molecules, but today’s researchers must operate at or beyond the utmost limits of conventional methods. In February, 1984 (1) we reported on a new approach to precolumn derivatization and analysis of amino acids which appeared to satisfy all the needs of protein chemists. The approach was based upon the formation of a phenylthiocarbamyl (PTC) derivative of the amino acids (2). The method was shown to be rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The method allows for the rapid, bonded phase separation with ultraviolet (UV) detection at 254 nm of the common amino acids with 12 minute analysis time and a one picomole sensitivity for a standard amino acid sample.

Amino Acid Analysis of Submicrogram Hydrolyzate Samples

We have recently reported1–3 on a new methodology for amino acid analysis (AAA) that relies on the reaction of free amino acids with Edman’s reagent, phenylisothiocyanate (PITC), to form the phenylthiocarbamyl (PTC) derivatives, which are subsequently separated and quantitated using a reverse phase gradient liquid chromatography (LC) system for analysis. This work extended the methodology for AAA using PITC introduced several years ago by Tarr and co-workers4–5, and appeared concurrently with a report by Heinrikson and Meredith6 describing similar methodology for AAA. The method was shown to be reliable, accurate, and extremely rapid (12 minute) with a maximum one picomole detection limit for each amino acid. This new method provides improved capabilities in comparison with conventional ion-exchange, post-column ninhydrin reaction systems. In addition, linear response in the 1020131000 pmol range was demonstrated as well as a high degree of reproducibility1. This system has been recently commercialized under the name PICO·TAG™ Amino Acid Analysis System3.

Thomas L. Tarvin

Papers

Rapid analysis of amino acids using pre-column derivatization.

PITC derivatives in amino acid analysis

A new, rapid, high-sensitivity analysis of amino acids in food type samples.

Amino Acid Analysis of Submicrogram Hydrolyzate Samples

Amino Acid Analysis Using Pre-Column Derivatization with Phenylisothiocyanate: Matrix Effects and Tryptophan Analysis