Rapid Production of Functionalized Recombinant Proteins:  Marrying Ligation Independent Cloning and in Vitro Protein Ligation

2006 ◽  
Vol 17 (3) ◽  
pp. 610-617 ◽  
Author(s):  
Susanna Kushnir ◽  
Yoann Marsac ◽  
Reinhard Breitling ◽  
Igor Granovsky ◽  
Vera Brok-Volchanskaya ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Protein Ligation ◽  
Ligation Independent Cloning ◽  
Rapid Production ◽  
Vitro Protein

scholarly journals In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5185 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Sara Züger ◽  
Edith Buchinger ◽  
Hideo Iwaï
Keyword(s):  
Protein Ligation ◽  
Naturally Occurring ◽  
Vitro Protein

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals In vitro protein digestibility of enzymatically pre-treated cocoa bean powder using commercial protease

2021 ◽  
Vol 828 (1) ◽  
pp. 012024
Author(s):  
W Haliza ◽  
E Y Purwani ◽  
D Fardiaz ◽  
M T Suhartono
Keyword(s):  
Protein Digestibility ◽  
Cocoa Bean ◽  
In Vitro Protein Digestibility ◽  
Vitro Protein

In Vitro Protein Degradation of 38 Sainfoin Accessions and Its Relationship to Tannin Content by Different Assays

2012 ◽  
Vol 60 (20) ◽  
pp. 5071-5075 ◽  
Author(s):  
Martin M. Lorenz ◽  
Christine Hayot Carbonero ◽  
Lydia Smith ◽  
Peter Udén
Keyword(s):  
Protein Degradation ◽  
Tannin Content ◽  
Vitro Protein

Cell-free, protein-synthesizing system of photosynthetic and heterotrophic Rhodospirillum rubrum

1976 ◽  
Vol 22 (2) ◽  
pp. 304-308
Author(s):  
C. T. Chow
Keyword(s):  
Protein Synthesis ◽  
Protease Activity ◽  
Rhodospirillum Rubrum ◽  
Chemical Compounds ◽  
Protein Synthesis Inhibitors ◽  
Free Protein ◽  
Two Systems ◽  
The Difference ◽  
Vitro Protein

An active in vitro protein-synthesizing system has been developed from Rhodospirillum rubrum grown under either photosynthetic or heterotrophic conditions. A protease activity has been found in both of these systems, and this activity can be readily inactivated by treating the cells with KCl and phenylmethyl sulfonylfluoride. The difference in protein-synthesizing activity between the photosynthetic and the heterotrophic systems has been tested in regard to the requirement of various chemicals and the response to protein synthesis inhibitors or various chemical compounds. It has been concluded that only minor differences in protein-synthesizing activity exist between these two systems.

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