Table 2.

Examples of factors that have compromised protein integrity for use in drug discovery projects, resulting observations and actions taken to overcome the issues.

Protein target	Quality control issue	Biophysical methods employed	Observations	Actions taken
Lactate dehydrogenase	Cofactor present in protein preparation	ITC, SPR	Tool compounds and added cofactor binding more weakly than expected	New purification method established
ATAD2	Protein aggregation	NMR, ITC, TSA	Protein showing poor spectrum, negative shifts with compounds in TSA, no binding of tool compounds	New construct designed
ACPER	Reduced binding functionality	ITC	Low stoichiometry and enthalpy for cofactor binding	New batch of protein prepared
MAPKAPK2	No binding to p38	NMR, ITC	Short construct used for NMR did not show binding to p38 and differences in compound affinity observed for long and short constructs in phosphorylation assays	Longer construct, containing putative site for p38a binding, used for activity and mechanistic assays

Where ATAD2 is ATPase family AAA domain-containing protein 2, ACPER is acyl carrier protein enoyl reductase, MAPKAPK2 is MAPK activated protein kinase 2.