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Patrick Lo, Ph.D. and Kristie Nybo, Ph.D.
BioTechniques, Vol. 53, No. 4, October 2012, p. 205
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It's denature of the beast

Equilibrium and kinetic analysis of protein denaturation using heat or a chaotrope such as urea in combination with fluorescence or circular dichroism spectroscopy is a standard approach to monitor protein unfolding. However, such techniques typically require substantial amounts of pure protein and depend on fairly elaborate and costly instrumentation. In contrast, differential scanning fluorimetry (DSF) is capable of monitoring the thermal denaturation of small amounts of protein in the presence of a hydrophobic fluorescent dye (such as SYPRO Orange) using only a modified real-time PCR thermocycler. SYPRO Orange fluoresces strongly upon binding the exposed internal hydrophobic domains of target proteins that result from thermal denaturation. In DSF, the fluorescence of SYPRO Orange in the presence of a protein is monitored as a function of increasing temperature, and the resulting curve is used to derive the Tm. This technique, however, cannot be used to analyze the kinetics of protein unfolding. In this issue, K. Storey and colleagues at Carleton University (Ottawa, Canada) describe their modification of DSF that enables analysis of protein unfolding kinetics in real-time using urea or other chaotropes over a constant or gradient temperature. The authors used their modified DSF method to examine the denaturation kinetics of lysozyme and hexokinase in the presence of increasing concentrations of urea. At each urea concentration, SYPRO Orange fluorescence was measured over time, and a line fitted by linear regression to the initial portion of the denaturation curve was used to derive the rate of denaturation. When denaturation rates were plotted against urea concentration, it was possible to determine the half-maximal and maximal rates of protein denaturation, as well as the cooperativity of individual denaturants in protein unfolding. By taking advantage of small sample volumes, 96-well PCR microplates, short procedure times, and with the ability to examine synergistic temperature effects, this method allows researchers to easily combine numerous experimental variables together to analyze the unfolding kinetics of proteins.

See “Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler”.

A sticky situation

Large-scale production of recombinant proteins is required for many biotechnology applications, including protein crystallization for structural studies. Several systems for protein production are available, including bacterial, yeast, baculoviral, and cell-free systems. However, for therapeutic applications and crystallography studies, proteins must be produced in mammalian expression systems to maintain proper folding and modifications. In these cases, transient transfection is an attractive approach for introducing a recombinant gene of interest, but protein yields from such methods are often inadequate, leaving researchers with the challenge of establishing stable cell lines through laborious selection procedures when higher protein quantities are necessary. Seeking a simpler selection method for stable protein-producing cell clones, Ojala et al. from the Karolinska Institute (Stockholm, Sweden) turned to SCARA5, a class A member of the scavenger receptor family that had previously been shown to promote cell adhesion to serum-coated tissue culture dishes. This characteristic was of particular interest since Ojala's group was laboring to produce stable HEK-293/EBNA cells producing a recombinant laminin α3 chain fragment (LN3G), which when expressed, led to significantly reduced adhesion of isolated clones and cell death. Co-expression of full-length SCARA5 in LN3G-producing cells allowed successful culturing of LN3G-transfected cells, with double transfectants showing higher adhesion and viability than cells expressing LN3G alone. Based on these initial experiments, the authors went on to develop a new antibiotic-free selection method for establishing stable cell clones using a vector with SCARA5 expressed on the same mRNA as a protein of interest by inclusion of an IRES sequence. The method showed a 40- to 60-fold increase in the number of protein-expressing cells following the wash steps and proved suitable for stable line selection in CHO and HEK-293 cells, the two most common mammalian cell lines used for production of recombinant proteins. The adhesive property of SCARA5 provided selective pressure toward high-expressing clones, eliminating the requirements for staining and sorting, and extending the ability to produce proteins with adverse effects on host cell adhesion and viability.

See “A novel scavenger receptor 5-based antibiotic-independent selection method for generation of stable recombinant protein-producing mammalian cell lines especially suitable for proteins affecting cell adhesion”.