Production of Recombinant Gelonin Using an Automated Liquid Chromatography System

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05–0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Download Full-text

Characterization of epitopes of seminal plasma antigen stimulating human monoclonal sperm-immobilizing antibodies: a personal review

Reproduction Fertility and Development ◽

10.1071/rd9890193 ◽

1989 ◽

Vol 1 (3) ◽

pp. 193 ◽

Cited By ~ 10

Author(s):

S Isojima

Keyword(s):

Monoclonal Antibody ◽

Recombinant Dna ◽

Human Monoclonal Antibody ◽

Human Sperm ◽

Biologically Active ◽

Blood Type ◽

Type I ◽

Recombinant Dna Technology ◽

Sperm Binding ◽

Carbohydrate Epitopes

In observations made between 1974 and 1984, 40 of 303 women with unexplained sterility (13.2%) showed positive sperm-immobilizing antibodies. Among many kinds of antibodies to human sperm including sperm coating antigens, the biologically active antibodies, such as sperm-immobilizing agglutinating antibodies and blocking antibodies for fertilization, could be relevant to infertility. Harmless sperm-binding antibodies are present even in the sera and cervical mucus of fertile women. Antigens corresponding to monoclonal antibodies (1C4, 2C6, 2E5), which were generated to human sperm coating antigens and indicated strong sperm-immobilizing activities, seemed to have carbohydrate epitopes. The majority of women with sterility of unknown cause appeared to raise sperm-immobilizing antibodies to carbohydrate epitopes of sperm. The stable human-mouse heterohybridoma H6-3C4 secreting monoclonal antibody (IgM, lambda) with extremely high titres of sperm-immobilizing (SI50, 5000 units) and agglutinating (1:1600) activities was successfully established from peripheral lymphocytes of a sterile woman. The chemical structure of an antigen epitope corresponding to human monoclonal antibody H6-3C4 was found to consist of internally repetitive, unbranched N-acetyllactosamine (blood type i antigen). Ejaculated human sperm appeared to be densely covered with sialyl blood type i antigen and sialyl branched internally repetitive N-acetyllactosamine (sialyl blood type I antigen). The antibody-producing V-H and V-L genes of the human hybridoma H6-3C1 were cloned and preserved to stabilize antibody production. Class-switch variants of heavy chain from mu to gamma (IgG1, lambda) in human Mab H6-3C4 were produced by recombinant DNA technology.

Download Full-text

Large scale high-performance liquid chromatography of urogastrone produced by recombinant DNA technology

Journal of Chromatography A ◽

10.1016/s0021-9673(01)87000-x ◽

1986 ◽

Vol 362 ◽

pp. 443-449 ◽

Cited By ~ 9

Author(s):

Stephen J. Brewer ◽

Charles H. Dickerson ◽

Jonathan Ewbank ◽

Anthony Fallon

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

Large Scale ◽

Recombinant Dna ◽

Recombinant Dna Technology ◽

Dna Technology

Download Full-text

Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

Revista de Chimie ◽

10.37358/rc.08.11.2004 ◽

2008 ◽

Vol 59 (11) ◽

Cited By ~ 1

Author(s):

Iulia Lupan ◽

Sergiu Chira ◽

Maria Chiriac ◽

Nicolae Palibroda ◽

Octavian Popescu

Keyword(s):

Amino Acids ◽

Aspartic Acid ◽

Enzymatic Synthesis ◽

Large Scale ◽

Recombinant Dna ◽

Industrial Enzymes ◽

Recombinant Dna Technology ◽

Bacterial Fermentation ◽

Natural Protein ◽

Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

Download Full-text

Protein-Engineered Polymers Functionalized with Antimicrobial Peptides for the Development of Active Surfaces

Applied Sciences ◽

10.3390/app11125352 ◽

2021 ◽

Vol 11 (12) ◽

pp. 5352

Author(s):

Ana Margarida Pereira ◽

Diana Gomes ◽

André da Costa ◽

Simoni Campos Dias ◽

Margarida Casal ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Recombinant Dna ◽

Biological Properties ◽

Resistant Bacteria ◽

Recombinant Dna Technology ◽

Free Standing ◽

Microbial Cell Factories ◽

Cell Factories ◽

Antimicrobial Materials

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

Download Full-text

Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽

10.3390/pharmaceutics12060549 ◽

2020 ◽

Vol 12 (6) ◽

pp. 549

Author(s):

Laura Garcia-Perez ◽

Anita Ordas ◽

Kirsten Canté-Barrett ◽

Pauline Meij ◽

Karin Pike-Overzet ◽

...

Keyword(s):

Gene Therapy ◽

Severe Combined Immunodeficiency ◽

Good Manufacturing Practice ◽

Proof Of Concept ◽

Preclinical Development ◽

Hematopoietic Stem ◽

Immune Deficiencies ◽

Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Download Full-text