Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants

Abstract

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

Targeted Tumor Therapy With a Fusion Protein of an Antiangiogenic Human Recombinant scFv and Yeast Cytosine Deaminase

“…By means of the fusion protein A33CDy that contains a scFv specific for human glycoprotein A33, we recently demonstrated reproducible highyield protein expression. 21 On the basis of these results we FIGURE 5. Immunotherapy with L19CDy-His.…”

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

“…Somewhat higher values could in some instances be obtained with yeasts: For S. cerevisiae, yields between 4 μg and 4 mg of scFv-GFP per liter of culture medium were reported [6]. The group of Markus Deckert developed a Pichia pastoris-based secretory production system for an anti-A33 (colorectal carcinoma antigen) A33scFv::GFP fusion protein and reached yields up to 12 mg/L [10,11]. For S. pombe, there is one report on the successful production of a biologically functional scFv-GFP fusion protein, but in that case, the fusion protein was not secreted [1].…”

“…Since its fluorescence property is preserved in chimeric fusion proteins, GFP has become a very popular reporter tag for specific scFv antibody fragments. There are many reports describing the expression of scFv-GFP fusions, which can be used for tumor assessment, drug screening, and for studying intracellular localization and dynamics of gene products in both in vitro and in vivo biological systems [1][2][3][4][5][6][7][8][9][10][11][12][13]. Although many applications make use of fluorescently labeled antibody fragments for immunological detection purposes, direct conjugation of scFvs with autofluorescent GFP can also be used for visualization and quantification.…”

“…Yeast expression systems combine some of the advantages of mammalian and bacterial cultures in providing a eukaryotic cell system that can be handled in a similar way as bacteria, allowing for economic production of proteins, and several yeast species have been described as well suited expression systems for scFv-GFP fusions [1,6,10,11]. Apart from protein production as such, secretion of the recombinantly expressed proteins is generally advantageous since protein purification from a culture supernatant is considerably more convenient than from a total cell lysate.…”

A visualized fusion protein based on self-assembly hydrophobin HGFI

“…When HGFI-GFP with a proper structure assembled on siliconlized glass and PCL surface, HGFI used its hydrophobic patch to adhere to hydrophobic substrate, and made the hydrophilic patch toward outside. For the hydrophilic mica, HGFI could bind the mica surfaces via the hydrophilic protein domainto increase the hydrophobicity of mica surface [28][29][30] . Poly ε-caprolactone(PCL) is considered as an environmentally friendly and valuable material for tissue engineering and drug delivery application, for it is nontoxic, cyto-compatible and bio-degradable in nature [31,32] .…”