Enhancing recombinant protein quality and yield by protein stability profiling.
The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.
Mezzasalma
Tara M
TM
Johnson & Johnson Pharmaceutical Research & Development, LLC, Exton, Pennsylvania 19341, USA.
Kranz
James K
JK
Chan
Winnie
W
Struble
Geoffrey T
GT
Schalk-Hihi
Céline
C
Deckman
Ingrid C
IC
Springer
Barry A
BA
Todd
Matthew J
MJ
D002021:Buffers; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D009994:Osmolar Concentration; D020836:Protein Structure, Quaternary / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000592:standards; D012492:Salts / Q000494:pharmacology; D012996:Solutions / Q000494:pharmacology; D013816:Thermodynamics
D002021:Buffers; D008670:Metals; D011994:Recombinant Proteins; D012492:Salts; D012996:Solutions; D016186:Receptor, Macrophage Colony-Stimulating Factor; D051057:Proto-Oncogene Proteins c-akt