Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Abstract

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔG RNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

“…In a previous study, highly expressed recombinant GFP with the hydrophilic N-termini ME 6 (hy, +1.82) and MK 6 (hy, +1.82), referred to as ME 6 -GFP and MK 6 -GFP, respectively [1], had an “artificial” C-terminal peptide, LeuGlu (the corresponding peptide for the Xho I restriction site; referred to as “LE” below)-6×His (His tag; referred to as “6H” below), not specified in the previous study [1], derived from the expression vector pET22b(+) (Additional file 1: Table S3, clones 3 and 5). Thus, we constructed clones to remove the corresponding nucleotide sequence of LE-6H (6H; 6 aa; hy, –0.28), including from the control clone GFP-LE-6H-Stop(TGA) (Additional file 1: Table S3, clone 1), but leaving the native hydrophilic C-terminal end sequence of GFP (MDELYK; 6 aa; hy, +0.35) and native stop codon (TAA).…”

“…The total and soluble expression levels of the GFP and ME 6 -GFP proteins from the deleted clones GFP-Stop(TAA)-# and ME 6 -GFP-Stop(TAA)-# were increased to 20.0 and 19.9%, and 17.6 and 27.7%, respectively, compared to those of the undeleted controls (Figure  1, lanes 2 and 4). These results indicate that when the C-terminal end is more hydrophilic, specificity for the largest translocon (i.e., the Tat channel) was increased compared to that of the hydrophilic N-terminus [1]. The increased total protein expression level suggests that the increased Tat pathway specificity helped to synthesize protein in the cytoplasm by a type of non-feedback regulation, secreting the synthesized protein quickly into the periplasm.…”

“…Previously, we characterized the N-terminal specific Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for periplasmic soluble expression of unfolded and folded target proteins [1]. Using green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting an isoelectric point (pI) and hydrophilicity separately, with an anchor sequence, M(X)(Y)[pI]-anchor-8×Arg(hydrophilicity)-GFP [pI and hydrophilicity separately], the bulky, folded protein was able to pass efficiently through the Tat pathway via the translocon with the largest diameter; moreover, passage was controlled by the pI value of the N-terminus in the order of acidic > neutral.…”

“…In this study, based on the confirmed result that the hydrophilic N-terminus attached to GFP had the Tat pathway specificity shown previously [1], we aimed to identify the hydrophilic role of the native C-terminal end of GFP (MDELYK; 6 aa; hydrophilicity [hy], +0.35) for soluble expression through the Tat pathway. We constructed the corresponding clones after deleting the LeuGlu(LE)-6×His (6H; 6 aa; hy, –0.28) peptide, derived from pET22b(+), and evaluated the total and soluble GFP expression levels.…”

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli

“…Various functional peptides, recombinant vaccines, and catalytic enzymes could be displayed on bacterial surfaces through appropriate surface anchors, which facilitated their further engineering and applications for certain purposes [ 2 ]. In gram-negative bacteria, two major secretion systems, the general secretion (Sec) and twin arginine translocation (Tat) pathways [ 3 – 4 ], have been identified to transport proteins to the extracellular milieu under the guidance of various N-terminal signal peptides. Most surface carriers in bacteria are surface presenting proteins that use an N-terminal signal sequence to guide their translocation to the cell surface, including OmpA, Omp85, and LamB [ 5 – 6 ].…”