A multi-column plate adapter provides an economical and versatile high-throughput protein purification system

Abstract

Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

How a highly acidic SH3 domain folds in the absence of its charged peptide target

“…dendrobatidis (AbpBD) had the following sequence: MGYREATAVYDYVAAEPNELSFNEGNLITHIVFVSEDWWQGTLNGVVGLFPGNYVELK QLEHHHHHH. For equilibrium and chemical denaturation assays, the proteins were expressed in autoinduction media and Ni-NTA, followed by purification under denaturing conditions as previously described (34). Proteins were further purified using ion-exchange with a HiTrap-Q column (Cytiva) to >95 % purity and dialysed in 10 mM Tris, pH 8.1 buffer.…”

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows

“…15 ), SG scientists modified these systems (sometimes using existing laboratory equipment like peristaltic pumps) to improve their functionality [3] , [4] , [5] , [6] . This type of work is continued to this day by scientists, as well as ÄKTA engineers that have developed newer versions [7] , [8] , [9] . While multiple FPLC systems could be run in parallel, this is an expensive alternative to manually processing several protein purifications by gravity column.…”

How a highly acidic SH3 domain folds in the absence of its charged peptide target

“…A pET32b-based (Novagen) plasmid containing the AbpSH3 domain from from S. cerevisiae (AbpSC) with a C-terminal his-tag had the following sequence: MAP-KENPWATAEYDYDAAEDNELTFVENDKIINIEFVDDD WWLGELEKDGSKGLFPSNYVSLGNLEHHHHHH and the AbpSH3 from the distant ortholog B.dendrobatidis (AbpBD) had the following sequence: MGYREATAVY-DYVAAEPNELSFNEGNLITHIVFVSEDWWQGTLNGVV GLFPGNYVELKQLEHHHHHH. For equilibrium and chemical denaturation assays, the proteins were expressed in autoinduction media and Ni-NTA, followed by purification under denaturing conditions as previously described (Dominguez et al, 2018). Proteins were further purified using ion-exchange with a HiTrap-Q column (Cytiva) to >95% purity and dialzsed in 10 mM Tris, pH 8.1 buffer.…”