Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6]. Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only. Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author

Identification of a novel RPGR mutation associated with X-linked cone-rod dystrophy in a Chinese family

Background Cone-rod dystrophy (CORD) is a group of inherited retinal dystrophies, characterized by decreased visual acuity, color vision defects, photophobia, and decreased sensitivity in the central visual field. Our study has identified a novel pathogenic variant associated with X-linked cone-rod dystrophy (XLCORD) in a Chinese family. Methods All six family members, including the proband, affected siblings, cousins and female carriers, have underwent thorough ophthalmic examinations. The whole exome sequencing was performed for the proband, followed by Sanger sequencing for spilt-sample validation. A mammalian expression vector (AAV-MCS) with mutated retinitis pigmentosa GTPase regulator (RPGR) sequence was expressed in HEK293 T cells. The mutated protein was verified by Western blotting and immunohistochemistry. Results A novel mutation in the RPGR gene (c.2383G > T, p.E795X) is identified to be responsible for CORD pathogenesis. Conclusions Our findings have expanded the spectrum of CORD-associated mutations in RPGR gene and serve as a basis for genetic diagnosis for X-linked CORD.

Neuroinductive properties of mGDNF depend on the producer, E. Coli or human cells

The glial cell line‐derived neurotrophic factor (GDNF) is involved in the survival of dopaminergic neurons. Besides, GDNF can also induce axonal growth and creation of new functional synapses. GDNF potential is promising for translation to treat diseases associated with neuronal death: neurodegenerative disorders, ischemic stroke, and cerebral or spinal cord damages. Unproductive clinical trials of GDNF for Parkinson’s disease treatment have induced to study this failure. A reason could be due to irrelevant producer cells that cannot perform the required post-translational modifications. The biological activity of recombinant mGDNF produced by E. coli have been compared with mGDNF produced by human cells HEK293. mGDNF variants were tested with PC12 cells, rat embryonic spinal ganglion cells, and SH-SY5Y human neuroblastoma cells in vitro as well as with a mouse model of the Parkinson’s disease in vivo. Both in vitro and in vivo the best neuro-inductive ability belongs to mGDNF produced by HEK293 cells. Keywords: GDNF, neural differentiation, bacterial and mammalian expression systems, cell cultures, model of Parkinson’s disease.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants

A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. Here described is a simplified and inexpensive Golden Gate Cloning Protocol in which the amplified PCR fragments that enter the one-step-one-pot reaction are stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The protocol inludes the design step, in which fragments are designed to posses unique overhangs, amplification of modules using a high fidelity polimerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) in one tube, assembly, DpnI digestion for eliminating the background plasmids that remain after the PCR reaction and transformation of the resulting assembly reaction into competent E.coli cells. The protocol eliminates the need for vectors and inserts. The plasmid is build using only PCR products or fragments, with one of them containing the bacterial origin of replication and the antibiotic resitance gene for selection. Multiple modular plasmids can be constructed in just one day with minimal hands-on time.

Plants as alternative platforms for monoclonal antibodies productions.

Monoclonal antibodies (mAbs) are widely used in medical therapy and diagnostics, veterinary therapy, and research. The demand for mAbs reaches several dozen tons per year and is constantly growing, approaching the limits of current production possibilities. Mammalian expression systems, which currently dominate the bioproduction industry, have limited production capacity and require high capital investment and production costs. Plants are becoming promising expression platforms due to their scalability, speed, low cost of production, low risk of contamination from animal pathogens and eukaryotic mechanisms of post-translational protein modification. The transgenic plants used for the production of mAbs can be obtained by stable transformation of plant cells as well as transient expression of foreign proteins. In this review, we extract a broad overview of articles, many of them from recent years, concerning modern approaches to producing monoclonal antibodies in plants, methods for modifying the carbohydrate profile of mAbs, and purifying the resulting product. We also present current data on the practical use of mAbs in medical therapies and potential methods of producing antibodies on a very large scale, able to meet the future market demand.