Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH) in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2) is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

Background. Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis in patients with congenital hypothyroidism (CH). In this study, the prevalence of TPO gene mutations in patients with thyroid dyshormonogenesis in Isfahan was investigated. Methods. In this cross-sectional study, genomic DNA of 41 patients with permanent CH due to thyroid dyshormonogenesis was extracted using the salting out method. The 17 exonic regions of the TPO gene were amplified. SSCP technique was performed for scanning of the exonic regions of the TPO gene, except exon 8. DNA sequencing was performed for those with different migration patterns in SSCP by chain termination method. Exon 8 was sequenced directly in all patients. In 4 patients, all fragments were also sequenced. Results. One missense mutation c.2669G > A (NM_000547.5) at exon 15 (14th coding exon) in one patient in homozygous form and seven different single nucleotide polymorphisms (SNPs) in exons 1, 7, 8, 11, and 15 of TPO gene. Conclusion. The TPO gene mutations among CH patients with dyshormonogenesis in Isfahan were less frequent in comparison with other similar studies. It may be due to the presence of other unknown gene mutations which could not be detected by SSCP and sequencing methods.

Background:

Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.

Methods:

A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr− cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.

Results:

The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.

Discussion:

This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZαA vector under control of AOX1 promoter, downstream of the secretion-α-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS-PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems.