Ligation-independent cloning is a simple method that provides several advantages over conventional cloning. However, the efficiency of ligation-independent cloning is considerably lower than that of conventional methods. Several studies have shown that competent cells used for ligation-independent cloning should preferably have a transformation efficiency of 106-107 cfu/μg DNA. Although such levels can be easily achieved using standard protocols with most Escherichia coli strains, some strains attain mush lower values. When such strains have to be used for ligation-independent cloning, certain measures need to be taken to avoid any situation that may further decrease the efficiency of the process. These measures, however, are usually time-consuming. This problem is exacerbated by the fact that some strains such as BL21 (DE3) appear to be intrinsically unsuited for ligation-independent cloning. Here we suggest that by avoiding DNA damage during purification simply by replacing UV transilluminators with blue light systems BL21 (DE3) cells with a transformation efficiency of 105 cfu/μg DNA can satisfactorily be used for ligation-independent cloning without any additional steps.

HIGHLIGHTS
•Using blue light instead of UV light increases the efficiency of LIC.
•A simple LED blue light projector combined with a filter can be used for this purpose.
•This approach is not recommended for applications that require higher sensitivity.

Amidolytic Activity of Factor VII Expressed in Iranian Lizard Leishmania

Elaheh Dianati, Mojgan Bandehpour, Mehrdad Hashemi, Bahram Kazemi

Trends in Peptide and Protein Sciences, Vol. 3 (2018), 1 June 2018 , Page 1-5 (e2)
https://doi.org/10.22037/tpps.v3i0.20924

Over many years, variable gene expression systems have been used obtaining of Factor VII protein, but each one has certain limitations. Therefore, the main goal of this study was to assess the biological activity of purified and recombinant factor VII expressed in Iranian lizard Leishmania. After transferring recombinant construct containing FVII gene to Leishmania, first, the expression of 55 kDa FVII protein in transfected cells was confirmed by analyzing cell lysate using SDS-PAGE and Western Blotting techniques. Then, FVII was purified by NI-NTA-His Tag- resin through an affinity chromatography. The chromogenic activity of human recombinant Factor VII (amidolytic activity) in supernatant and pellet fractions of Leishmania promastigotes was done using ELISA method. The amidolytic activity of rhFVII was about 0.0125 IU/ml in the concentrated cell sediment and 0 IU/ml in the supernatant in the first 60 minutes and after that time, none of the samples showed acceptable activity.

 

HIGHLIGHTS
• Factor VII has important role in the treatment of coagulation and hemophilic disease.
• Recombinant FVII was expressed in Iranian Lizard Leishmania.
• The amidolytic activity of rhFVII was about 0.0125 IU/ml in cell pellet.
• No amildolytic activity was observed in the supernatant.

Production Design of Efficient Recombinant Human Interleukin-4 (rhIL-4) under Specific Promoter in Escherichia coli

Mahmoud Vahidi, Mojgan Bandehpour, Bahram Kazemi

Trends in Peptide and Protein Sciences, Vol. 3 (2018), 1 June 2018 , Page 1-9 (e3)
https://doi.org/10.22037/tpps.v3i0.21996

Recombinant DNA technology plays a vital role in improving health conditions by developing new pharmaceuticals.  Recently, some of the cytokines as other recombinant proteins could be produced using the recombinant DNA technology. The role of Recombinant IL-4 in allergy, autoimmunity, and cancer has been investigated. The present study was aimed to clone and produce the Human IL-4 under specific promoter in Escherichia coli and assessed its biological functions. The designed hIL-4 gene construct was artificially synthesized; subsequently, it was sub cloned into the pcDNA3.1 (+) vector in HindIII restriction enzyme site. Recombinant plasmid was transferred and expressed in BL21 cells. The rhIL-4 protein was evaluated by SDS-PAGE and Western blotting. It was purified by Ni-NTA affinity chromatography. The purified protein concentration and also accuracy were determined by ELISA. MTT assay was applied to evaluate the biological activity of rhIL-4 on the Erythroleukemic cell line proliferation. The rhIL-4 gene was successfully cloned and transformed into expression E. coli cells. As a result, a specific band was observed both on the SDS-PAGE and nitrocellulose membrane after Western blotting. The purified protein concentration was equal to 500 pg/ml. The MTT assay indicated that the exposed cells with rhIL-4 were proliferated in a dose dependent manner. The rhIL-4 gene under specific eukaryotic promoter was successfully cloned in the prokaryotic system and the transcription was carried out by T7 RNA polymerase. Therefore, mass production of IL-4 could be a great help in clinical trials and research studies. Additionally, prokaryotic system used in current work was less costly and less time-consuming.

HIGHLIGHTS

  • Interleukin-4 (IL-4) is a cytokine that regulates multiple biological functions.
  • The recombinant Human IL-4 was cloned and produced under specific promoter in Escherichia coli.
  • Through MTT assay, the exposed cells with rhIL-4 were proliferated in a dose dependent manner.

Biosynthesis and Characterization of an Anticoagulant Chitinase from Fermented Wheat Bran & Shrimp Shells’ Substratum

Haritha Meruvu, Hari Bhanu Teresa Meruvu

Trends in Peptide and Protein Sciences, Vol. 3 (2018), 1 June 2018 , Page 1-9 (e4)
https://doi.org/10.22037/tpps.v3i0.23165

Solid state fermentation (SSF) of wheat bran coupled with shrimp shellfish waste Citrobacter freundii str. nov. haritD11 was optimized conventionally (112.43 U/gds) and statistically (124.73 U/gds). Chitinase was purified 4.24-fold with 31% yield and specific activity of 64.87 U/mg protein. The purified chitinase had a specific activity of 64.87 U/mg with optimal activity at pH 9 and temperature 45 ºC. The enzyme was stable at 8.0–9.5 pH range with 90% stability and between 45 °C – 60 °C for 1 hour. The Km value of the Citrobacter freundii haritD11 purified chitinase with swollen chitin (substrate) is 7.53 mg/mL with a Vmax of 2.27 mmol h−1mL−1. The purified chitinase was halotolerant showing maximum activity and stability up to 9% Sodium chloride, it also possessed potential antifungal and anticoagulant activity. This is the first report to date elucidating the production of halotolerant chitinase from wheat bran supplemented with shrimp shellfish waste using Citrobacter freundii haritD11 with notable tolerance to heavy metal ions, its application as an antifungal and anticoagulant agent.

HIGHLIGHTS


•Optimization of solid-state fermentation of Citrobacter freundii str. nov. haritD11.
•The halotolerant chitinase was produced from wheat bran supplemented with shrimp shellfish waste.
•The enzyme had notable tolerance to heavy metal ions.
•Chitinase was purified 4.24-fold with 31% yield and specific activity of 64.87 U/mg.
•The enzyme can be applied as an antifungal and anticoagulant agent.

Preparation and Characterization of Desmopressin Peptide Attached Multi-Walled Carbon Nanotube

Mohammadreza Gholibeikian, Amirreza Arvaneh, Mohammad Hassan Houshdar Tehrani

Trends in Peptide and Protein Sciences, Vol. 3 (2018), 1 June 2018 , Page 1-8 (e5)
https://doi.org/10.22037/tpps.v3i0.23138

Desmopressin, a synthetic analogue of vasopressin, has many applications in medicine including diabetes insipidus, night bedwetting, and hemophillia A. In this work, the attachment of desmopressin to multi-walled carbon nanotubes (MWCNTs), functionalized by HNO3 and H2SO4 treatment was first used to remove the unwanted catalyst from MWCNTs and meanwhile introduced carboxylic acid groups onto the surface of MWCNTs. These carboxylic groups were then used as reaction sites for the attachment of desmopressin peptide to MWCNTs. The reagents used for the attachment were oxalyl chloride and dicyclohexylcarbodiimide (DCC). The Covalent attachment of desmopressin to functionalized MWCNTs was confirmed by Fourier Transform infrared spectroscopy (FT-IR), Raman scattering, and Field Emission Scanning Electron Microscopy (FESEM).

HIGHLIGHTS


•The conversion of carboxylic groups to oxalyl chloride on the surface of MWCNTs.
•The conversion of carboxylic groups to O-acylisourea on the surface of MWCNTs.
•DCC and oxalyl chloride activate groups on MWCNTs for nucleophilic substitution.
•Desmopressin is a cyclic nonapeptide with one disulfide bridge.
•The Covalent attachment of desmopressin to functionalized MWCNTs.

Selection of a suitable host strain is an important step in recombinant production of heterologous proteins. As the prokaryotic systems and especially Escherichia coli (E. coli) cells are the most attractive host in therapeutic protein production, various derivatives of this organism have been developed to overcome the limitations exist in recombinant protein production, such as inefficient expression and folding of target proteins with eukaryotic origin. This review summarized key strategies for E. coli engineering and introduced some new and applicable engineered E. coli strains that produce more complex proteins for therapeutic and research use in the future.

 

HIGHLIGHTS
• Selection of a suitable host is an important step in recombinant protein production.
• Escherichia coli cells are the most attractive host in therapeutic protein production.
• Engineered strains had developed to produce active protein with suitable conformation in industrial scale.
• Classical mutagenesis & genome engineering are two important strain engineering strategies.