Recently, several comparative studies have examined the effects of various fusion partners on total and soluble expression
yield (Table 2). Marblestone et al. evaluated the expression and solubility of three model proteins fused to the C termini
of MBP, GST, TRX, NusA, Ub, and SUMO tags.16
The tags were ranked in terms of increased total expression as TRX > SUMO ~ NusA > Ub ~ MBP ~ GST and increased soluble
expression as SUMO ~ NusA > Ub ~ GST ~ MBP ~ TRX. Hammarstrom et al. cloned 27 human proteins (MW < 20 kDa) into various expression
vectors and ranked the tags' ability to promote soluble expression as TRX ~ MBP ~ Gb1 > ZZ > NusA > GST > His6.17 Braun et al. compared the expression of 32 human proteins (molecular weight of which varied from 17 to 110 kDa) and ranked
tags in terms of increased expression and yield after purification as GST ~ MBP > CBP > HIS6.18 Shih et al., in a study of 40 different proteins with eight different tags, observed that MBP gave the best overall results
in terms of total and soluble expression.19 In one of the studies in Dyson et al., the solubility of 20 mammalian proteins was compared and the fusion tags were ranked
in terms of increased soluble expression as TRX ~ MBP > HIS10 > GST > GFP.20 De Marco et al. demonstrated that NusA was better
than GST at enhancing the solubility and stability of recombinant proteins.21
Table 2. Summary of several comparative studies that examine the effects of various fusion partners on total and soluble expression
The inconsistency of the data from these comparative studies only solidifies the statement that tools and optimal conditions
for each protein remain empirical and that no technology or reagent is a panacea. Nevertheless, it is likely that in the future
generalities about specific fusion tags may be made (e.g., for entire protein classes). As the comparative studies suggest,
gene fusion tag systems range dramatically in efficiency.
YIELD AND ACTIVITY FACTORS
Factors that influence yield and biological activity include: a) the affinity purification scheme; b) enhancement of recombinant
protein expression; c) protein folding and enhanced solubility; d) protection from degradation; e) size of the fusion tag;
and f) the specificity, efficiency, and site of cleavage. Herein lies a further discussion of these factors.
Affinity Purification Scheme
Protein fusion tags are often used to purify proteins from crude extracts. GST and MBP are two examples. Other protein fusion
systems, such as NusA, SUMO, Split SUMO, thioredoxin, and ubiquitin require an affinity tag, such as polyhistidine (HIS).
Numerous examples of affinity purification exist for fusion proteins, including nickel-nitriloacetic acid to isolate hexahistidine-fused
proteins,22 amylose to isolate MBP-fused proteins,23 and GSH-sepharose to isolate glutathione (Table 3).24 Successful purification schemes achieve high quality and quantity with inexpensive, high capacity resins and mild elution
conditions. For effective purification, high affinity between the fusion tag and resin is essential, but affinity must not
be too high, because harsh elution conditions can disrupt tertiary structure.
Table 3. Affinity tags influence protein expression yield and activity.