Replacement of Tyr62 by Trp in the designer protein Milk Bundle‐1 results in significant improvement of conformational stability

Abstract

Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content, having potential application in animal nutrition. One of the limitations we face in this endeavor is the achievement of stable proteins in spite of a highly biased amino acid content. We report here the synthesis and characterization of MB-1Trp, a protein with a tailored content in selected essential amino acids. The protein is a Tyr62-Trp mutant of the parent molecule MB-1 described earlier. The new protein is largely helical as per design, is well folded, and has a melting temperature of 55³C. Its resistance to proteolytic degradation compares to that of cytochrome c, a protein of similar size. Design strategy used for MB-1Trp is discussed with regards to its applicability toward the creation of efficient nutritional proteins. ß

Single‐point mutations at the surface of MB‐1Trp lead to important changes in its conformational properties

“…Substitution of Leu in position 19 and 68 by Glu on the template protein MB‐1Trp (31) was performed using ‘Altered Sites II mutagenesis kit’ (Promega, Madison, WI, USA) and ‘Site‐directed Mutagenesis kit’ (Stratagene, La Jolla, CA, USA), respectively. The mutagenic oligonucleotides are shown below with the corresponding MB‐1Trp sequence (oligo 1 for position 19 and oligo 2 and 3 for position 68).…”

“…Measurements were done as described previously (31). Cytochrome c (ICN Biomedicals #101467) was used as an internal reference in order to allow comparison of different digestion experiments.…”

A Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Protein Ladder Made of Disulfide-Bridged Proteins

“…The protein used as the monomer (or repeating unit) is a mutant of the designer protein MB-1 (1) where the mutations Met10-Cys, Leu91-Cys, and Tyr62-Trp were performed by oligonucleotide site-directed mutagenesis (2). The production by Escherichia coli and purification protocol were carried out as reported in Morrison et al (3), except for few modifications.…”

“…Isolation of fulllength cDNA sequences that are rarely expressed is difficult because a highly specific enrichment technique is required. The presence of a large number of uncharacterized cDNA fragments in the form of expression sequence tags (EST) 2 (1) or DNA fragments as results of differential display (2) or other differential analysis (2-9) generates the need for an efficient method for obtaining the full-length cDNA through the information of a partial cDNA sequence. Although many methods have been developed to achieve this goal, obtaining a full-length cDNA sequence based on a small fragment of cDNA sequence remains a challenge.…”

“…E-mail: luoj@msx.upmc.edu. 2 Abbreviations used: EST, expression sequence tag; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; LACE, linear amplification of cDNA ends; HPRT, hypoxanthine phosphoribose transferase; DTT, dithiothreitol.…”