Targeting Optimal Buffers for Downstream Crystallisation Screening

The process of X-ray crystallography requires purified protein to form crystals in optimal buffer conditions on specialised crystallisation plates. A primary buffer screen of larger global parameters (buffer system, pH, salt, concentration etc.) before this experimental step can greatly reduce the window of buffers to test and minimise consumable costs. Proteins that easily aggregate or show instability can have their conditions optimised from a preliminary buffer screen before crystallisation trials. The stability of proteins in initial buffer conditions has been directly linked with crystal formation success in subsequent crystallography screens [1]. One method of measuring protein stability in a buffer screen is with differential scanning fluorimetry (DSF). Comparison of thermal melting profiles of a protein in different buffers can indicate the conditions that increase protein stability. We show how the SUPR-DSF can be used to rapidly screen bovine Beta-lactoglobulin (BLG) in a variety of buffer conditions to increase stability and subsequently aid in improving homogeneity in solution (Figure 1). The system’s 384-well plate format allows simple screening of commercially available buffer screens, plus replicates, in one thermal ramp experiment. These screens have minimal set-up time and require no extrinsic dyes. A preliminary buffer screen can be used to ensure proteins are stored in optimal buffer conditions prior to any structural methodologies being implemented.

Conclusion
Beta-Lactoglobulin is the most abundant protein found in bovine milk. It has undergone many stability and folding studies due to its importance in the dairy industry. Its physiological role is yet to be defined though it is hypothesised to be involved in the transport of molecules between parent and offspring [4]. The confirmation and stability of BLG has been linked greatly with altered pH conditions and buffer composition [5]. BLG notably alters oligomeric state at differing pHs, whether its monomeric or dimeric form affects stability would require further research approaches [6]. Our screen showed increased stability of BLG in not only citric acid but also low pH SPG, indicative of a preference for acidic buffers to maintain stability. The results shown corroborate with the published literature of BLG, whereby structure has been elucidated successfully in high salt acidic buffer screens, specifically, 3 M NaCl buffered at pH3.8 with sodium citrate [2]. The stability of proteins in an initial buffer condition screen has been directly linked with crystal formation success [1]. The DSF data reported shows success in being able to narrow down conditions before crystallisation screening trials. This confirmation of results outlines SUPR-DSF as a suitable high throughput system to rapidly screen optimal buffer conditions for recombinant proteins. As many commercial buffer screens come in 96-well format, the SUPR-DSF 384-well format allows replicate screening within the same experimental set up. SUPR-DSF is a suitable system for buffer screening before downstream structural methods and as a primary screen before setting up of crystallography plates. The need to not add extrinsic dyes also aids in elucidation of true protein solubility results. Buffer screening for protein homogeneity should be considered an integral part of protein purification research.