Immune Netw.  2010 Apr;10(2):35-45. 10.4110/in.2010.10.2.35.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

• Hur BU
• Choi HJ
• Yoon JB
• Cha SH

• KMID: 2150658
• DOI: http://doi.org/10.4110/in.2010.10.2.35

Abstract

BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research.
METHODS
We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain.
RESULTS
The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly.
CONCLUSION
We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

Keyword

Fv; Leucine zipper; Recombinant antibody; Alkaline phosphatase; DsbC

MeSH Terms

Alkaline Phosphatase
Antibodies
Clone Cells
Cytoplasm
DNA
Escherichia
Escherichia coli
Immunoglobulin Fragments
Indicators and Reagents
Leucine Zippers
Oxidoreductases
Pathology, Molecular
Pyruvic Acid
Sprains and Strains
Alkaline Phosphatase
Antibodies
DNA
Immunoglobulin Fragments
Indicators and Reagents
Oxidoreductases
Pyruvic Acid

Figure

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  Figure 1 Schematic diagram depicting the cytoplasmic zipFv expression vectors used in this study.

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  Figure 2 ELISA to prove antigen-binding specificity of zipFv-112 and its fusions with alkaline phosphatase produced in the cytoplasm of Origami(DE3) cells. Origami(DE3) cells transformed with pCzFv-112, pCzFvHAP-112 or pCzFvLAP-112 were grown in the presence of 0.1 mM IPTG, and the presence of PDC-E2-specific zipFv antibody fragments (zipFv-112, zipFv-112(H-AP) or zipFv-112(L-AP)) in the cytoplasmic extracts was determined by ELISA. Mouse anti-myc tag mAb was used for a primary antibody as described under Materials and Methods. MBP, IL-15 and BSA were used as negative control antigens, and TMB substrate was used to visualize signals. Data represent the average of three experiments±standard deviation.

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  Figure 3 Comparison of antigen-binding reactivity of the zipFv-112 and its fusions with alkaline phosphatase produced in the cytoplasm of Origami(DE3) cells by ELISA using anti-myc mAb for detecting the VH-Fos-myc fragments. Origami(DE3) cells producing zipFv-112, zipFv-112(H-AP) and zipFv-112(L-AP) were grown in the presence of 0.1 mM IPTG, and the presence of PDC-E2-specific zipFv-AP antibody fragments in the serial dilutions of the cytoplasmic extracts was determined by ELISA using anti-myc mAb followed by goat anti-mouse IgG-HRPO conjugated for detecting the VH-Fos-myc or the VH-Fosmyc-AP fusions as described under Materials and Methods. TMB substrate was used to visualize signals. Data represent the average of three experiments±standard deviation.

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  Figure 4 Comparison of antigen-binding reactivity of anti-PDC-E2 zipFv fused with alkaline phosphatase by ELISA. Origami(DE3) cells producing the zipFv-112(H-AP) and the zipFv-112(L-AP) were grown in the presence of 0.1 mM IPTG, and the presence of PDC-E2-specific zipFv-AP antibody fragments in the serial dilutions of the cytoplasmic extracts was determined by ELISA using AP substrate, pNPP, for detecting AP activity of the VH-Fos-myc-AP or the VL-Jun-AP fusions as described under Materials and Methods. MBP was used as a negative control antigen. Data represent the average of three experiments±standard deviation.

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  Figure 5 Western blot showing the expression of the VH-Fos-myc, the VH-Fos-myc-AP or the VL-Jun-AP fragments of the zipFv-112, the zipFv-112(H-AP) and the zipFv-112(L-AP). Origami(DE3) cells expressing the zipFv-112, the zipFv-112(H-AP) and the zipFv-112(L-AP) were grown in 2× YT/amp medium supplemented with 0.1 mM IPTG, and total proteins in the cell lysates were separated by using 12% SDS-PAGE at reducing (A, lane 1 on B) and non-reducing (Lane 2 on B) condition. Western blot was performed using either mouse anti-myc tag mAb followed by goat anti-mouse IgG-AP conjugated or NBT/BCIP substrate directly as described under Materials and Methods to detect the presence of the VH-Fos-myc and the VH-Fos-myc-AP, or AP activity of the VH-Fos-myc-AP and the VL-Jun-AP polypeptides, respectively.

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  Figure 6 Comparison of antigen-binding reactivity of the zipFv-112(L-AP) and the zipFv-112(H-AP) produced in the cytoplasm of Origami(DE3) cells by ELISA using anti-myc mAb for detecting signals. Origami(DE3) cells producing zipFv-112(L-AP) and zipFv-112(H-AP) were grown in the presence of 0.1 mM IPTG, and presence of the functional zipFv-AP with PDC-E2 binding reactivity in the serial dilutions of the cytoplasmic extracts was determined by ELISA using mouse anti-myc mAb followed by goat anti-mouse IgG-HRPO conjugated for detecting the VH-Fos-myc or the VH-Fos-myc-AP fragments. Data represent the average of three experiments±standard deviation.

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  Figure 7 Effect of DsbC on the production of functional zipFv-112(H-AD/L-AP) determined by ELISA. Origami(DE3) cells carrying pCDF-DsbC were transformed with the pCzFvHADLAP-112, and grown in the presence of 0.1 mM IPTG. Presence of the functional zipFv-112(H-AD/L-AP) that bound to PDC-E2 with or without DsbC co-expression was determined using serial dilutions of the cytoplasmic extracts. (A) Mouse anti-myc mAb followed by goat anti-mouse IgG-HRPO conjugated or (B). pNPP, AP substrate, was used to detect antigen-binding signals in ELISA. Data represent the average of three experiments±standard deviation.

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