Blood Res.  2015 Jun;50(2):103-108. 10.5045/br.2015.50.2.103.

Human coagulation factor VIII domain-specific recombinant polypeptide expression

• Choi SJ
• Jang KJ
• Lim JA
• Kim HS

• KMID: 2172774
• DOI: http://doi.org/10.5045/br.2015.50.2.103

Abstract

BACKGROUND
Hemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII), the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners.
METHODS
To determine F8 domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography.
RESULTS
Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII.
CONCLUSION
Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.

Keyword

Hemophilia A; Coagulation factor VIII; Coagulation factor IX; Domain-specific recombinant FVIII; Hep3B hepatocytes

MeSH Terms

Antibodies
Bacteria
Blood Coagulation
Chromatography, Affinity
Clone Cells
Factor IX
Factor VIII*
Glutathione Transferase
Hemophilia A
Hemostasis
Hepatocytes
Humans
Peptides
Antibodies
Factor IX
Factor VIII
Glutathione Transferase
Peptides

Figure

• 

  Fig. 1 Cloning of each F8 domain. (A) A1, A2, B, A3, C1, and C2 represent the domains of human FVIII, and the bars indicate the cloning region of each domain. The numbers under each bar indicate the corresponding sequence of amino acids in each domain. (B) pET-28a(+) vectors containing each recombinant F8 domain were digested with BamHI and XhoI. Each domain was subcloned into pGEX4T-2, a GST expression vector. The plasmids were purified, and the products were subjected to BamHI and XhoI digestion. Lane 1, pGEX only; lane 2, pGEX-A1; lane 3, pGEX-A2; lane 4, pGEX-A3; and lane 5, pGEX-C. Arrowheads indicate the products of each domain. M1: 1 kb DNA marker, M2: 100 bp DNA marker.

• 

  Fig. 2 Expression of recombinant FVIII polypeptides tagged with GST (A, B) or His (C, D). (A) Expression of recombinant FVIII polypeptides was induced by incubation with 0.5 mM IPTG for 4 hr at 25℃. To assess expression, whole-cell lysates were separated in 10% polyacrylamide gels. Gels were stained with Coomassie Brilliant Blue. (B) Proteins in the gel were transferred to a PVDF membrane and immunoblotted with an anti-GST antibody. BL21: host bacterial strain, Mock: pGEX4T-2 vector introduced into BL21. (C) To express His-tagged polypeptides corresponding to FVIII domains, the DNA fragment of each domain was subcloned into pET28a, a His-tagging expression vector. Expression of the His-tagged, domain-specific polypeptides was induced with 1 mM IPTG. (D) His-tagged polypeptides corresponding to FVIII domains were identified by western blotting using an anti-His-tag antibody. Arrowheads in (A) and (C) indicate the corresponding domain polypeptides.

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  Fig. 3 Interaction of recombinant FVIII domain polypeptides with FIX. To characterize recombinant FVIII domain polypeptides, a pull-down assay was performed with FIX. GST-tagged polypeptides were purified and incubated with either recombinant FIX (A) or standard human plasma (B). The interacting proteins were precipitated by incubation with a 50% slurry of glutathione-agarose beads. The proteins bound to the GST-FVIII complexes were eluted and analyzed by western blotting with anti-GST (upper box) or anti-FIX (lower box) antibodies. Specific lanes: GST pull-down with GST-FVIII-A1 (lane 1), GST-FVIII-A2 (lane 2), GST-FVIII-A3 (lane 3), and GST-FVIII-C (lane 4).

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