Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein. To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography. The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons. Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.

Download Full-text

A Novel Hemagglutinin with Antiproliferative Activity against Tumor Cells from the Hallucinogenic MushroomBoletus speciosus

BioMed Research International ◽

10.1155/2014/340467 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Jian Sun ◽

Tzi-Bun Ng ◽

Hexiang Wang ◽

Guoqing Zhang

Keyword(s):

Cation Exchange ◽

Antiproliferative Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Lymphocytic Leukemia ◽

Hemagglutinating Activity ◽

Cation Exchange Chromatography ◽

Type Ab

Little was known about bioactive compounds from the hallucinogenic mushroomBoletus speciosus. In the present study, a hemagglutinin (BSH,B. speciosushemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg2+and slightly inhibited by Fe2+, Ca2+, and Pb2+. None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210)in vitro, with IC50of 4.7 μM and 7.0 μM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50of 7.1 μM.

Download Full-text

Process Engineering Strategy for Recombinant Protein Recovery from Canola by Cation Exchange Chromatography

Biotechnology Progress ◽

10.1021/bp980110y ◽

1999 ◽

Vol 15 (1) ◽

pp. 12-18 ◽

Cited By ~ 16

Author(s):

C. Zhang ◽

C.E. Glatz

Keyword(s):

Recombinant Protein ◽

Cation Exchange ◽

Process Engineering ◽

Exchange Chromatography ◽

Protein Recovery ◽

Cation Exchange Chromatography ◽

Engineering Strategy

Download Full-text

Purification of in vitro produced mouse monoclonal antibodies. A two-step procedure utilizing cation exchange chromatography and gel filtration

Journal of Immunological Methods ◽

10.1016/0022-1759(85)90395-3 ◽

1985 ◽

Vol 79 (1) ◽

pp. 89-98 ◽

Cited By ~ 42

Author(s):

Mats Carlsson ◽

Anders Hedin ◽

Mats Inganäs ◽

Bengt Härfast ◽

Fred Blomberg

Keyword(s):

Monoclonal Antibodies ◽

Cation Exchange ◽

Gel Filtration ◽

Exchange Chromatography ◽

Cation Exchange Chromatography ◽

Step Procedure

Download Full-text

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Microbial Cell Factories ◽

10.1186/s12934-021-01642-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ram Shankar ◽

Nina Schäffer ◽

Marco Schmeer ◽

Joe Max Risse ◽

Karl Friehs ◽

...

Keyword(s):

Escherichia Coli ◽

Cation Exchange ◽

Plasmid Dna ◽

Recombinant Expression ◽

Exchange Chromatography ◽

Dna Purification ◽

Specific Productivity ◽

Scale Production ◽

Cation Exchange Chromatography ◽

Plasmid Purification

Abstract Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms21030928 ◽

2020 ◽

Vol 21 (3) ◽

pp. 928 ◽

Cited By ~ 2

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.

Download Full-text

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography

Journal of Chromatography A ◽

10.1016/j.chroma.2014.12.057 ◽

2015 ◽

Vol 1379 ◽

pp. 74-82 ◽

Cited By ~ 2

Author(s):

Sandeep Vemula ◽

Akshay Dedaniya ◽

Rahul Thunuguntla ◽

Maheswara Reddy Mallu ◽

Pavani Parupudi ◽

...

Keyword(s):

Protein Folding ◽

Cation Exchange ◽

Exchange Chromatography ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Cation Exchange Chromatography ◽

In Vitro Refolding ◽

Stimulating Factor

Download Full-text

Expression and Purification of Tetanus Toxin Fragment C in Escherichia coli BL21(DE3)

Protein and Peptide Letters ◽

10.2174/0929866527666200528113327 ◽

2020 ◽

Vol 27 (11) ◽

pp. 1132-1140

Author(s):

Pengdi Chai ◽

Xiuying Pu ◽

Jianqiang Li ◽

Xiaoyu Xia ◽

Jun Ge ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Secondary Structure ◽

Cation Exchange ◽

Anion Exchange ◽

Tetanus Toxin ◽

Soluble Proteins ◽

Exchange Chromatography ◽

Cation Exchange Chromatography ◽

Simple Method

Background: Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine. Objective: In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems. Methods: The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice. Results: The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity. Conclusion: The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

Download Full-text

Generation of Mature Nα-Terminal Acetylated Thymosinα1 by Cleavage of Recombinant Prothymosinα

The Scientific World JOURNAL ◽

10.1155/2013/387282 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Bo Liu ◽

Xin Gong ◽

Shaohong Chang ◽

Peng Sun ◽

Jun Wu

Keyword(s):

Mass Spectrometry ◽

High Performance Liquid Chromatography ◽

High Performance ◽

Expression System ◽

Exchange Chromatography ◽

Cation Exchange Chromatography ◽

Reverse Phase ◽

E Coli ◽

Amino Acid Peptide

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. Nα-terminal acetylated thymosinα1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosinα(ProTα) inE. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTαwas cleaved by human legumain expressed inPichia pastoristo release Tα1in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of anE. coliexpression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides inE. coli.

Download Full-text