Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Stability, and Function

Short peptide tags attached to recombinant proteins are emerging as an important tool for biochemical research. Here, we report the effects of 10 Solubilization Controlling Peptide (SCP) tags on crystallization behavior of a bovine pancreatic trypsin inhibitor (BPTI) variant. The tags did not affect structure, thermodynamics, and activities of BPTI. Moreover, eight of the tagged variants crystallized under the same condition, and six of them diffracted at high resolution. All variants with long-term solubility (<i>LS</i>) between 1 and 6 mg/mL produced crystals that diffracted well, while variants with <i>LS</i> < 1 and >6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different condition. The only exception was a glutamine tagged variant, which had an <i>LS</i> of 5 mg/mL, but fast aggregation kinetics, and produced mere needles unsuitable for further analysis. Crystal structures indicated that most tags were largely invisible, indicating high flexibility, without having interactions with nearby residues. Therefore, short peptides, introducing a mere 5–7 residue elongation, could provide a useful technology for tuning protein solubility without affecting its other properties and hence for overcoming problems associated with excessively low or high solubility, such as in crystallization

MOESM1 of The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant

Additional file 1: Figure S1. Secreted protein by transformant of PC-3-7 and QM9414. A: Avicel cultivation; 10 µL of the supernatant from day 5 of PC-3-7 series and day 6 of QM9414 series were subjected to SDS-PAGE. Gels were stained by Coomassie Brilliant Blue. B: Cellobiose cultivation; 20 µL of supernatant from day 2.5 of PC-3-7 series and day 3 of QM9414 series were subjected to SDS-PAGE. Gel of Avicel cultivation was stained by Coomassie Brilliant Blue and that of cellobiose cultivation was silver stained.   Figure S2. Gene expression profile of PC-3-7 transformants on Avicel induction. Genes analyzed were cbh1 (A), egl1 (B), bgl2 (C), and xyr1 (D). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray bar indicates PC-∆bgl2. Values represent the relative expression of each gene normalized to act1 as an internal control. Values represent the means of triplicate experiments. Error bars indicate standard deviations.   Figure S3. Effect of bgl2 mutation and disruption in T. reesei QM9414. A: specific activity of the intracellular cellobiase from transformants QM9414, QM-Mbgl2 and QM-∆bgl2. Cellobiase activity is derived from the mean of triplicate experiments. Error bars indicate standard deviation. B: HPLC analysis of transglycosylation products using cell-free extracts from transformants. Details are as in Fig. 2. Putative transglycosylation products are indicated by arrows.   Figure S4. Southern analysis of transformants for bgl2 analysis. A: the schematic representation of genomic structure of each transformants. B: results of hybridization by gene specific probe for bgl2 and pyr4. Genomic DNA of each transformants was digested by SacI. Lane M represents molecular marker. Table S1. PCR primers used for plasmid construction. Table S2. primers used for real-time quantitative PCR