Production Of Native Proteins Research Articles

Boosting the enzymatic activity of CxxC motif-containing PDI family members.

Compounds harboring high acidity and oxidizability of thiol groups permit tuning the redox equilibrium constants of CxxC sites of members of the protein disulphide isomerase (PDI) family and thus can be used to accelerate folding processes and increase the production of native proteins by minimal loading in comparison to glutathione.

THE IMPACT OF THE EUROPEAN GREEN DEAL STRATEGY ON SECURITY IN THE SUPPLY OF NATIVE PLANT PROTEIN FOR ANIMAL FEED IN POLAND

This article aims to indicate the potential impact of the European Green Deal strategy’s solutions on the production of native protein crops for animal feed and its role in achieving sovereignty in the supply of this raw material in Poland. Self-sufficiency in the supply of plant protein is a concern that has been recognized and widely discussed in the national and European scientific community among practitioners involved in the production of livestock feed and, above all, among politicians of the European Commission, which decides the final shape of the EU Common Agricultural Policy. The strategy adopted by the European Commission, referred to as the European Green Deal, proposes restrictions on the use of plant protection products and mineral fertilizers. This will not take place without having an impact on production and the economic situation in agriculture. The research was conducted based on Statistics Poland data and the results of scientific studies. It was concluded that the implementation of the proposed strategy could be a factor that activates the production of native leguminous plants due to their nitrogen-fixing properties. Consequently, this may contribute to an increase in the production of native plant protein for animal feed, and thus to a greater sovereignty in the supply of this raw material for feed purposes.

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

AbstractBACKGROUNDRecombinant proteins produced for use as biopharmaceuticals need to harbor their native N‐terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion‐tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N‐terminus.RESULTSHuman fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed‐batch cultivations. The protein of interest (POI) carried an N‐terminal affinity fusion‐tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion‐tag with a circularly permuted human caspase‐2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N‐terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 μg mL−1 dsDNA and ~ 1000 EU mL−1 endotoxin. Only a small number of E. coli host cell proteins were co‐purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off‐target cleavage could be observed.CONCLUSIONOur findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion‐protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2‐based platform fusion protein process (CASPON technology) purification platform. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Fused Split Inteins: Tools for Introducing Multiple Protein Modifications.

The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

A novel self-cleavage system for production of soluble recombinant protein in Escherichia coli

Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications.

Development of an Insect Cell-Free System

Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out post-translational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications.

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.

Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli

To directly express native recombinant proteins in Escherichia coli, a new expression vector pSB was constructed using Ssp DnaB mini-intein. Using the vector, native proteins could be produced with the help of C-terminal self-cleavage of the intein. In this study, we cloned hIFNα-4 gene into pSB and used E. coli strain Origami B (DE3) as the host. Expression experiments were carried out both in Shake flasks and a 5 L bioreactor. The results indicated hIFNα-4 could be expressed in the form of soluble protein with correct folding in E. coli. The maximal hIFNα-4 content was 21.7% of total protein, and the antiviral activity of the protein was 1.2 × 10 8 IU mg −1. Overall, good effects were achieved with this system. This intein-mediated protein expression system opens up a useful method for production of native recombinant protein in E. coli.

Protein topology affects the appearance of intermediates during the folding of proteins with a flavodoxin-like fold

The topology of a native protein influences the rate with which it is formed, but does topology affect the appearance of folding intermediates and their specific role in kinetic folding as well? This question is addressed by comparing the folding data recently obtained on apoflavodoxin from Azotobacter vinelandii with those available on all three other α–β parallel proteins the kinetic folding mechanism of which has been studied, i.e. Anabaena apoflavodoxin, Fusarium solani pisi cutinase and CheY. Two kinetic folding intermediates, one on-pathway and the other off-pathway, seem to be present during the folding of proteins with an α–β parallel, also called flavodoxin-like, topology. The on-pathway intermediate lies on a direct route from the unfolded to the native state of the protein involved. The off-pathway intermediate needs to unfold to allow the production of native protein. Available simulation data of the folding of CheY show the involvement of two intermediates with characteristics that resemble those of the two intermediates experimentally observed. Apparently, protein topology governs the appearance and kinetic roles of protein folding intermediates during the folding of proteins that have a flavodoxin-like fold.

New Strategies in Polypeptide and Antibody Synthesis: An Overview

The synthesis of radioligands can benefit considerably from optimized recombinant protein production, both on the aspect of economy of production and on the level of improving the targeting and pharmacokinetics of the ligand. This paper first describes a general production optimization strategy, and then elaborates on a protein design strategy tailored to targeting applications. Production in Escherichia coli will benefit from economy of goods and time as compared to other organisms. In order to increase the chance of finding a successful production system in this host, we have assembled a large number of expression strategies in a single, uniform expression system (FastScreen). The system allows rapid optimization of direct production of native proteins or via a fusion protein strategy with subsequent recovery of the desired protein. As an example of recombinant radioligand synthesis for improved targeting and clearing, a manifold of intermediate molecular size was synthesized by fusing one Fab and two single-chain variable fragments (scFv) antibody binding fragments into a trifunctional molecule (Tribody). Due to the use of the specific heterodimerization of the Fab chains, trispecific, bispecific, or trivalent antibody derived targeting reagents can easily be obtained. Recombinant production techniques also allow for specific incorporation of amino acids favoring a site specific labeling (labeling tags).

Recombinant production of native proteins from Escherichia coli.

The production of large quantities of proteins became possible with the advent of recombinant DNA technology, and the subsequent expression of recombinant proteins in Escherichia coli (E. coli) (Itakura, 1977). Recombinant proteins are produced in E. coli cytoplasm at high concentrations in a very different environment than that in which they are normally expressed. Their structure, stability and solubility are affected by this environment and can be quite different than that of the native protein. Therefore, purification of recombinant proteins often requires different techniques than those used to purify the same protein from its natural environment. Numerous attempts have been made to purify and/or refold expressed proteins.

Conformational toxicity and sporadic conformational diseases

Spontaneous, so-called ‘conformational’ diseases, specially of the neurodegenerative type like Alzheimer's, are linked to certain protein types which have the normal amino-acid sequence but are misfolded and accumulate due to resistance to proteolysis. In the case of prion diseases, the ‘protein only’ hypothesis assumes that the misconformation of a native protein could be initiated upon interaction with a sister-protein already in the misfolded state. There is an alternative to this sister protein contamination scheme, which assumes that the misconformation is acquired upon protein synthesis, that is de novo. Misfoldling and resistance to proteolysis could result from defects responsible for shortage or inactivity of the cellular factors in charge of protein folding and degradation. The defects could have a genetic origin (the gene of the faulty factor involved could have been mutated, or control and regulation of its expression could have been altered, etc.). Alternatively, the cell's actual biosynthetic and/or proteolytic resources could have become overloaded and unavailable, due to unscheduled mass-production of proteins resulting from unscheduled cell growth or proliferation, cell stress, etc. Xenobiotics, active for instance as endocrine proliferators, stressors, or inducing copious, unscheduled gene expression, etc. could give rise to shortage of cellular factors necessary for the production of native proteins and for proteolysis. Alternatively, xenobiotics could alter expression or activity of some of these factors. In both cases, the xenobiotic could be a ‘conformational toxicant’ by inducing misfolding of selected proteins. The xenobiotic could trigger some conformational disease if it targets a specific protein and tissue.