Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA

Academia Sinica, Beijing, China.
BioTechniques (Impact Factor: 2.95). 12/2003; 35(5):1032-8, 1041-2.
Source: PubMed


Overproduction of genetically engineered antibodies, such as single-chain antibodies (scAbs) in Escherichia coli often results in insoluble and inactive products known as inclusion bodies. We now report that fusion or co-expression of FkpA, the E. coli periplasmic peptidyl-prolyl-isomerase with chaperone activity, substantially improves soluble and functional expression of scAbs. Anti-human bladder carcinoma scAb (PG) and anti-human CD3 x anti-human ovarian carcinoma-bispecific scAb (BH1) were fused with FkpA on the pTMF-based plasmid and expressed in E. coli. More than half of the amount of each expressed fusion protein FkpA-PG or FkpA-BH1 was soluble. In addition, the fusion protein cellulose-binding domain from Cellulomonas fimi (CBD)-PG and anti-human CD3 x anti-human CD28 x anti-human ovarian carcinoma-trispecific scAb (TRI) fused to the pelB (a signal peptide from pectate lysase B of a Bacillus sp.) signal sequence were co-expressed with FkpA under the control of the T7 promoter. A substantial portion of the co-expressed CBD-PG or TRI was soluble. Furthermore, PG, BH1, and TRI were biologically active as judged by ELISA and in vitro cytotoxicity assay. These results suggest that overexpression of FkpA should be useful in expressing heterologous proteins in E. coli.

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Available from: Zhong Zhang, Aug 26, 2015
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    • "The expression of FkpA alleviated the RpoE-dependant stress response in E. coli cells during accumulation of misfolded proteins [14] and it also suppressed the formation of inclusion bodies and promoted proper folding when co-expressed with a folding-defective protein variant [15]. The co-expression of FkpA with scFv significantly improved the latter's soluble and functional expression [16]. Although these protein folding factors are increasingly exploited to improve the soluble expression of recombinant protein products in the periplasm, the detailed impact on host cell metabolism is still not clearly understood. "
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    ABSTRACT: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression. The periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes. The simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.
    Microbial Cell Factories 04/2010; 9(1):22. DOI:10.1186/1475-2859-9-22 · 4.22 Impact Factor
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    • "SurA, also a parvulin, was found to improve the folding of unstable or aggregation-prone proteins in the periplasm [176] but failed to help production of a scFv fragment [173]. Meanwhile FkpA, which like trigger factor possesses both chaperone and PPIase activity, enhanced production of a wide range of scFv fragments by up to 10-fold when overproduced [173], while its fusion to various scAb fragments also led to increased solubility and higher functional yields [177]. FkpA co-production also led to increased hydrolysis of ampicillin by a catalytic scFv [178] and enhanced the production of penicillin acylase [179]. "
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    ABSTRACT: Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed - and largely unpredictable - results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies.
    Microbial Cell Factories 02/2009; 8(1):9. DOI:10.1186/1475-2859-8-9 · 4.22 Impact Factor
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    • ") or Western blot analysis (data not shown). To explore its soluble expression, various factors were examined, including the optical density of culture at induction (OD 600 nm 0.4–0.8), the concentration of inducer IPTG (0.1–1.0 mM), induction temperature (37, 25, 16, and 12 °C) [16] [17] [18] as well as co-expression with molecular chaperone GroESL [19]. Unfortunately, no significant amount of the soluble target product was observed under any of the above conditions . "
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    ABSTRACT: An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.
    Protein Expression and Purification 11/2004; 37(2):361-7. DOI:10.1016/j.pep.2004.06.037 · 1.70 Impact Factor
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