Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Abstract

The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.

Expression, Immobilization and Enzymatic Properties of Glutamate Decarboxylase Fused to a Cellulose-Binding Domain

“…N -terminal amino acid sequence analysis confirmed that proteolytic cleavage occurred in linker region (-ATTM↓STTT-, ↓ indicates cleavage site). This phenomenon might be related to the fact that inter-domain linkers are particularly susceptible to proteolysis [12]. The reason is that flexible regions can more easily adopt conformations compatible with the active site of endopeptidase [12].…”

“…EcGAD possesses some superior enzymatic characteristics including a high affinity for substrate and high reaction rate compared to other GADs [1,4]. Some studies have focused on potential for industrial applications of EcGAD by improving its enzymatic properties and reaction conditions [6,9,12]. However, few studies have been performed on the immobilization of EcGAD for reuse [7]; in particular, there is no report of experiments using an affinity tag.…”

Coupled selection of protein solubility in E. coli using uroporphyrinogen III methyltransferase as red fluorescent reporter

“…It is identified that different linkers affect soluble expression and stability of some fusion proteins (Kavoosi et al, 2007). In this study, two different linkers were independently incorporated between the maize protein and reporter, but their effect on improving protein expression was limited.…”

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

“…Flexible linkers are typically incorporated in cases where the fused domains require a certain degree of movement or interaction (Kim et al, 2000;Blazyk and Lippard, 2004;Jiang et al, 2005;Wang et al, 2007;Lu and Feng, 2008). Most commonly used flexible linker sequences primarily consist of Gly and Ser residues ("GS" linker) (Huston et al, 1988;Kavoosi et al, 2007;Hölsch and Weuster-Botz, 2010). (G 4 S 1 ) n is one such widely used sequence.…”