Protein Expression and Purification

E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins...

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of 19 F nuclei, 19 F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in V...

We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column...

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops...

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product...

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the S-protein. Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD...

Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed Pycnoporus sanguineus CDH (psCDH) on the surface of Bacillus subtilis spores for the first time. Enzymatic characterization revealed that spore surface display enhanced the tolerance of psCDH to high temperature (80 °C) and low pH levels (3...

Diaminopropionate ammonia-lyase transforms D and L isomers of 2,3-diaminopropionate to pyruvate and ammonia. It catalyzes D- and l-serine less effectively. L-2,3-diaminopropionate is a precursor in the biosynthesis of oxalyl diaminopropionate as a neurotoxin in certain legume species. In this work, we cyclized the diaminopropionate ammonia-lyase from Salmonella typhimurium in vitro using the redox-responsive split intein, and identified that backbone cyclization afforded the enzyme with the improved activity, thermal stability and resistance to the exopeptidase proteolysis, different from effects of the incorporated sequence recognized by tobacco vein mottling virus protease at C-terminus...

Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this study, a method for the expression and purification of anti-TSLP nanobody (Nb3341) was established at 7 L scale and subsequently scaled up to 100 L scale...

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads...

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat...

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site...

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step...

Natural ginsenoside needs to be converted into rare ginsenoside before it can be readily absorbed into the bloodstream for action. In this study, an α-l-arabinofuranosidase (α-l-AFase) gene Bsafs2 was cloned from Bacillus subtilis (B. subtilis). Bsafs2 was ligated to the expression vector pET28a(+), and the expression vector was constructed and transformed into Escherichia coli (E. coli) BL21 heterologous recombinant expression to obtain α-l-AFase. α-l-AFase can hydrolyze at the C20 site of Ginsenoside Rc to obtain rare ginsenoside Rd...

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM)...

Human pepsinogens (mainly pepsinogen I and pepsinogen II) are the major inactive precursor forms of the digestive enzyme pepsin which play a crucial role in protein digestion. The levels and ratios of human pepsinogens have demonstrated potential as diagnostic biomarkers for gastrointestinal diseases, particularly gastric cancer. Nanobodies are promising tools for the treatment and diagnosis of diseases, owing to their unique recognition properties. In this study, recombinant human pepsinogens proteins were expressed and purified as immunized antigens...

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC)...

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through...

A lipase EstA from Bacillus subtilis KM-BS was expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme achieved high activity (49.67 U/mL) with protein concentration of 1.29 mg/mL under optimal conditions at the large-scale expression of 6 h and post-induction time at 30 °C using 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The optimal temperature and pH of the purified enzyme were at 45-55 °C and pH 8.0 - 9.0, respectively. Activity of the purified enzyme was stable in the presence of 1 mM Ca2+ ; stimulated by 1 mM Mg2+ and Mn2+ , and inhibited by Fe3+ ...

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world...