SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

Thie, Holger; Schirrmann, Thomas; Paschke, Matthias; Dübel, Stefan; Hust, Michael

doi:10.1016/j.nbt.2008.01.001

Cited by 54 publications

(43 citation statements)

References 44 publications

(52 reference statements)

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“…MalE, PelB, or TorT signal peptides were divided into two groups by their secretion pathways. MalE and PelB signal peptides lead their fusion proteins to the Sec-dependent pathway, and TorT signal peptide leads its fusion protein to the signal recognition particle pathway for translocation into the periplasm (Thie et al 2008). Our results suggested that expression of soluble aglycosylated rhFcγRI was possible via both secretion pathways of E. coli.…”

Section: Discussionmentioning

confidence: 66%

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli

Hatayama

Asaoka

Hoya

et al. 2012

Appl Microbiol Biotechnol

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Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K D =[1.7±0.2]×10−10 M) and IgG3 (K D=[1.1±0.2]×10−10 M) were similar to those of glycosylated rhFcγRI.

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Section: Discussionmentioning

confidence: 66%

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli

Hatayama

Asaoka

Hoya

et al. 2012

Appl Microbiol Biotechnol

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“…Our results showed that the mature cutinase was partially located in the periplasm, while some was secreted into the culture medium (14). Secretion into the culture medium was not unexpected, since similar phenomena have been observed in the expression of other secretory proteins (15)(16)(17)(18)(19). After optimization of the cultivation conditions in shake flasks, the cutinase activity in the culture medium reached 149.2 U/ml (326 mg/liter) 52 h postinduction (14).…”

mentioning

confidence: 64%

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Woodard

Chen

et al. 2013

Appl Environ Microbiol

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Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase NS ) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase NS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase NS S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase NS S130A, the cells expressing cutinase NS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase NS " was proposed to explain the underlying mechanism for the extracellular release of cutinase NS . Cutinase not only catalyzes the cleavage of the ester bonds of cutin, but is also capable of hydrolyzing soluble esters, insoluble triglycerides, and a variety of polyesters (1). In addition to its hydrolytic capability, cutinase is also used in ester synthesis (2, 3) and transesterification (4). Therefore, as a multifunctional enzyme, cutinase has potential uses in the food, chemical, textile, and other industries (5).Enzymes with cutinase activity have been found in both fungi and bacteria, such as Fusarium solani pisi and Thermobifida fusca, respectively (5-7). It has been demonstrated that cutinases from both groups belong to the ␣/␤-hydrolase superfamily, share similar spatial structures, and display similar catalytic properties, as well as substrate specificities. In addition, consistent with their ability to accommodate large substrates like cutin, they contain an open active site, and the lid structure normally seen in most lipases is absent in cutinase. Despite these similarities, there does not appear to be significant sequence homology between fungal and bacterial cutinases; thus, it has been suggested that they be classified into prokaryotic and eukaryotic cutinase subfamilies, respectively (6).To date, all the studied cutinases from microorganisms have been found to be secretory enzymes (6). Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11, 12), and Pichia pastoris (13). The highest yield of extracellu...

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“…Because the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm, the mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasm membrane could be a key determinant for the folding and, consequently, for the expression of the displayed protein on the phage surface (25)(26)(27)(28)(29)(30)(31). Alternative sequences in the signal peptide region have been known to modulate the expression level and folding quality of the displayed protein (25,27,31), but difficulty remains in identifying optimum signal sequences in a vast sequence space for some sc-dsFv constructs.…”

Section: Discussionmentioning

confidence: 99%

“…The key discovery is that the signal sequence variants of the sc-dsFv-pIII fusion protein have varying effects in directing the sc-dsFv expression on the recombinant phage surface. The signal sequence has been known to be responsible for the Sec system-dependent translocation of the pIII fusion protein from the translation site in the cytoplasm to the periplasm membrane (25)(26)(27)(28)(29)(30)(31), a critical process for the integration of the displayed protein on the recombinant phage surface (32). But the optimal signal sequences for the translocation of the pIII fusion protein were not known.…”

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confidence: 99%

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Huang

Chen

et al. 2010

Journal of Biological Chemistry

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Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and scdsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv. Single chain variable fragment (scFv)2 displayed as a fusion protein amino-terminal to the pIII minor capsid protein on the filamentous phage surface is one of the most prominent methods in antibody engineering. The small size of the scFv construct enables superior tissue-penetrating capabilities over the whole IgG or Fab fragment (1), making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (for reviews see Refs. 2-8). scFv is a single polypeptide chain antibody fragment construct encoding a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains (9 -11). The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. Moreover, an scFv library, which could contain more than one billion scFv variants, can be propagated with an Escherichia coli vector of bacterial phage origin (12, 13); the recombinant phages displaying the scFv variants can be selected or screened for antigen binding and re-amplified with the host E. coli. The sequences of the selected scFv can be further diversified with various mutagenesis technologies for affinity maturation (14, 15). As such, the scFv scaffold supports a powerful in vitro technology mimicking the production of high affinity antibodies in mammalian immune responses to foreign antigens, and the re...

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SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

Cited by 54 publications

References 44 publications

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

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