scholarly journals Recombinant Expression and Biophysical Characterization of a Druggable Schistosoma mansoni Universal Stress G4LZI3 Protein

2021 ◽  
Author(s):  
Abiola Fatimah Adenowo ◽  
Priscilla Masamba ◽  
Ndibonani Kebonang Qokoyi ◽  
Babatunji Emmanuel Oyinloye ◽  
Abidemi Paul Kappo
Keyword(s):  
Secondary Structure ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Recombinant Expression ◽  
Stress Protein ◽  
Size Exclusion ◽  
Appreciable Amount ◽  
Heterologous Protein Expression ◽  
Structural Characterisation ◽  
Restriction Digest

Purpose: Universal stress protein from S. mansoni, designated as G4LZI3, was previously hypothesised as a druggable target and vaccine candidate for human schistosomiasis. The purpose of this study is to characterize a purified recombinant G4LZI3 preliminarily for subsequent structural characterisation, which will provide baseline structural data for future functional studies for the discovery, design and development of new schistosomal drugs for the treatment, control and elimination of schistosomiasis. Methods: Restriction digest analysis of a GenScript-synthesised codon-optimised G4LZI3 gene construct was carried out to ascertain its integrity and size. Thereafter, the pQE30-G4LZI3 construct was transformed into an M15 bacterial expression host. Transformed cells were induced with isopropyl β-D-thiogalactoside for recombinant protein expression of an appreciable amount of pQE30-G4LZI3, which was subsequently purified with fast protein liquid chromatography and a size exclusion chromatographic purification scheme. Preliminary biophysical characterisation of the 6X His-tagged G4LZI3 was done to determine its secondary structure characteristics and protein stability. Results: A molecular weight protein of 20.3 kDa was confirmed subsequent to restriction digest analysis, while heterologous protein expression yielded a highly soluble and considerable amount of histidine-tagged G4LZI3 protein, which was successfully purified to homogeneity. Biophysical characterisation indicated that the protein was well folded, heat-stable, had the functional groups and secondary structure composition required and was thus amenable to further structural characterisation and determination. Conclusion: Biophysical characterisation of purified G4LZI3 showed that further structural studies can be embarked upon on the use of G4LZI3 as a druggable target and possibly a vaccine target against schistosomiasis via vaccinomics.

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

scholarly journals Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12497
Author(s):  
Fei Shang ◽  
Hui Wang ◽  
Dan Zhang ◽  
Wenhui Wang ◽  
Jiangliu Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Promoter Regions ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Pet Expression System

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

Recombinant protein expression and purification of Taq DNA polymerase v1

2021 ◽  
Author(s):  
Maira Rivera ◽  
Javiera Reyes ◽  
Javiera A Avilés ◽  
Amparo Núñez ◽  
Fernan Federici ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Dna Polymerase ◽  
Recombinant Protein Expression ◽  
Recombinant Expression ◽  
Storage Conditions ◽  
Expression And Purification ◽  
Taq Dna Polymerase ◽  
Protein Expression And Purification

This is a slightly modified and simplified version of a protocol by Thomas G.W. Graham et al, which is available at https://gitlab.com/tjian-darzacq-lab/bearmix and has been described in depth in the article 10.1371/journal.pone.0246647, for the recombinant expression of a E602D mutant of Taq DNA polymerase in pET-28a that is available in Addgene (Addgene plasmid # 166944 ; http://n2t.net/addgene:166944 ; RRID:Addgene_166944). The main goal of this protocol is to eliminate the use of large volumes for dialysis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions.

A Comprehensive Guide to the Commercial Baculovirus Expression Vector Systems for Recombinant Protein Production

2020 ◽  
Vol 27 (6) ◽  
pp. 529-537 ◽  
Author(s):  
Vibhor Mishra
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Expression Vector ◽  
Recombinant Protein Expression ◽  
Scale Up ◽  
Vector System ◽  
Heterologous Protein Expression ◽  
Baculovirus Expression ◽  
Vector Systems ◽  
Baculovirus Expression Vector

The Baculovirus Expression Vector System (BEVS) is a workhorse for recombinant protein expression for over thirty-five years. Ever since it was first used to overexpress the human IFN-β protein, the system has been engineered and modified several times for quick and easy expression and scale-up of the recombinant proteins. Multiple gene assemblies performed on the baculovirus genome using synthetic biology methods lead to optimized overexpression of the multiprotein complexes. Nowadays, several commercially available BEVS platforms offer a variety of customizable features, and often it is confusing which one to choose for a novice user. This short review is intended to be a one-stop guide to the commercially available baculovirus technology for heterologous protein expression in the insect cells, which users can refer to choose from popular and desirable BEVS products or services.

scholarly journals Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

2020 ◽  
Vol 21 (3) ◽  
pp. 928 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yulia A. Logashina ◽  
Yaroslav A. Andreev
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Reaction Medium ◽  
Cellular Metabolism ◽  
Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

scholarly journals The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Zou ◽  
Sha Li ◽  
Nan Li ◽  
Shi-Long Ruan ◽  
Jing Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Emission ◽  
Soluble Expression ◽  
Heterologous Protein Expression ◽  
E Coli

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

scholarly journals Choice of selectable marker affects recombinant protein expression in cells and exosomes

2021 ◽  
pp. 100838
Author(s):  
Chenxu Guo ◽  
Francis K. Fordjour ◽  
Shang Jui Tsai ◽  
James C. Morrell ◽  
Stephen J. Gould
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Selectable Marker ◽  
Recombinant Protein Expression ◽  
In Cells
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