Table 3. In Vitro and In Silico Methods for Protein Solubility Enhancement and Evaluation.

category	methodology/tool name
Expression condition optimization	Reducing speed, lowering temperature, optimizing growth media.100
Protein design based approaches	Site-directed mutagenesis, directed evolution, rational redesign on lipid exposed residues,208,226,254,262 rational design based on segment swapping,239,412 QTY Code,274 encoding gene modification.407
Protein fusion based approaches	Protein partner fusion: SIMPLEx,267 ribosomal protein,434 IDP;435 Solubility enhancing fusion tags: Mal-bp,416 GST,418 TRX,418 SlyD,419 DsbA,421 NusA,91 T7PK,421 Skp,428 HaloTag7,420 CBDengD,426 SEP(C9R),424,425 MOCR;455 Molecular chaperone fusion: DnaK,431 GroEL,430 RS-mTEV,432 Spy.433
Charges-mediated enhancement	negGFP.438
Surface hydrophobic residue/patch identification	Experimental: hydrophobic dye or tracer;46In silico prediction: SAP,48 hPatch.49
Protein solubility prediction	GraphSol,456 ProGan,457 DeepSol,96 PaRSnIP,458 Protein-Sol,93 Cam-Sol,94 PON-Sol,459 ESPRESSO,460 PROSO II,461 CCSOL,462,463 SCM,464 Samak-Wang,465 SOLpro,92 PRSP,466 SoDoPE.95