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Cell-free expression of natively folded hydrophobins
Publication date: Available online 4 February 2020Source: Protein Expression and PurificationAuthor(s): Rezwan Siddiquee, Samuel Choi, Shirley Lam, Patrick Wang, Ruhu Qi, Gottfried Otting, Margaret Sunde, Ann KwanAbstractHydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide b...
Source: Protein Expression and Purification - February 5, 2020 Category: Biochemistry Source Type: research

Skimmed milk as an alternative for IPTG in induction of recombinant protein expression
In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombi...
Source: Protein Expression and Purification - February 5, 2020 Category: Biochemistry Source Type: research

Expression, purification, and characterization of human mannose-6-phosphate receptor – Extra cellular domain from a stable cell line utilizing a small molecule biomimetic of the mannose-6-phosphate moiety
In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1–14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellul...
Source: Protein Expression and Purification - February 4, 2020 Category: Biochemistry Source Type: research

Obtaining active recombinant proteins from bacterial inclusion bodies using salt solutions under neutral pH conditions
Publication date: May 2020Source: Protein Expression and Purification, Volume 169Author(s): Marzieh Najafi, Yaghoub SafdariAbstractEukaryotic recombinant proteins expressed in bacterial cells usually aggregate within the cells as inclusion bodies. Despite the widely-accepted theory considering inclusion bodies as inactive materials, inclusion bodies may contain large amounts of correctly-folded active recombinant proteins. Proteins trapped in inclusion bodies can be released using a high pH solution (pH ≥ 11); however, they may undergo structural changes in such pH conditions that may lead to their inactivation. Shifti...
Source: Protein Expression and Purification - February 2, 2020 Category: Biochemistry Source Type: research

Bacterial expression, purification, and initial characterization of a full-length Cas13b protein from Porphyromonas gingivalis
Publication date: Available online 30 January 2020Source: Protein Expression and PurificationAuthor(s): Jiang Zhu, Xia Zhou, Xiaolan Huang, Zhihua DuAbstractThe CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (a...
Source: Protein Expression and Purification - January 31, 2020 Category: Biochemistry Source Type: research

Engineering Xaa-Pro dipeptidyl aminopeptidase for specific cleavage of glucagon and glucagon-like peptide 1 from fusion proteins
Publication date: Available online 30 January 2020Source: Protein Expression and PurificationAuthor(s): Erik Vernet, Marie Østergaard Pedersen, Henning Thøgersen, Allan Christian ShawAbstractN-terminal extensions (“tags”) have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered varia...
Source: Protein Expression and Purification - January 31, 2020 Category: Biochemistry Source Type: research

Heterologous production of porcine derived antimicrobial peptide PR-39 in Escherichia coli using SUMO and intein fusion systems
In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 μg PR-39/L from SUMO fusion system and 280 μg PR-39/L from the intein fusion system was achieve...
Source: Protein Expression and Purification - January 31, 2020 Category: Biochemistry Source Type: research

Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification
We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purifi...
Source: Protein Expression and Purification - January 29, 2020 Category: Biochemistry Source Type: research

Obtaining active single chain antibody from bacterial inclusion bodies using metal ions
Publication date: Available online 27 January 2020Source: Protein Expression and PurificationAuthor(s): Marzieh Najafi, Yaghoub SafdariAbstractEukaryotic recombinant proteins expressed in bacterial cells usually aggregate within the cells as inclusion bodies. Despite the widely-accepted theory considering inclusion bodies as inactive materials, inclusion bodies may contain large amounts of correctly-folded active recombinant proteins. Molecules trapped in inclusion bodies can be released using a high pH solution (pH ≥ 11); however, they may undergo structural changes in such pH conditions that lead to their protein i...
Source: Protein Expression and Purification - January 29, 2020 Category: Biochemistry Source Type: research

An optimized protocol for high yield expression and purification of an extremophilic protein
Publication date: Available online 25 January 2020Source: Protein Expression and PurificationAuthor(s): Mohamed N. Malash, Nahla A. Hussein, Shaden Muawia, Mahmoud I. Nasr, Rania Siam (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - January 26, 2020 Category: Biochemistry Source Type: research

Recombinant β-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries
Publication date: Available online 22 January 2020Source: Protein Expression and PurificationAuthor(s): R.L. Anisimov, O.A. Ershova, A.V. Ershov, M.A. Filatova, S.A. Katorkin, V.M. SimonovAbstractAntibodies specific to β-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein β-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific binders were discovered and converted to full-size hIgG1 antibodies leading to highly stable binders with dissocia...
Source: Protein Expression and Purification - January 23, 2020 Category: Biochemistry Source Type: research

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant
ConclusionLCL can be explored for its clinical potential. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - January 23, 2020 Category: Biochemistry Source Type: research

Editorial Board
Publication date: April 2020Source: Protein Expression and Purification, Volume 168Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - January 23, 2020 Category: Biochemistry Source Type: research

Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones
This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - January 22, 2020 Category: Biochemistry Source Type: research

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays
In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutat...
Source: Protein Expression and Purification - January 22, 2020 Category: Biochemistry Source Type: research