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.

Separation of Ionic Compounds by Reversed-Phase Liquid Chromatography An Update of Ion-Pairing Techniques

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

Separation of organic amine compounds on silica gel with reversed-phase eluents

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.

Brian A. Bidlingmeyer

Papers

Rapid analysis of amino acids using pre-column derivatization.

Separation of Ionic Compounds by Reversed-Phase Liquid Chromatography An Update of Ion-Pairing Techniques

PITC derivatives in amino acid analysis

Separation of organic amine compounds on silica gel with reversed-phase eluents

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