Chromatographic Techniques in the Downstream Processing of (Recombinant) Proteins

Author(s):  
Ruth Freitag
2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Nathaniel J. Kingsbury ◽  
Karen A. McDonald

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltratedNicotiana benthamianaleaves, up to 81% of a truncated version of E1 endoglucanase fromAcidothermus cellulolyticuswas recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins.


2015 ◽  
Vol 33 (6) ◽  
pp. 902-913 ◽  
Author(s):  
J.F. Buyel ◽  
R.M. Twyman ◽  
R. Fischer

2016 ◽  
Vol 120 ◽  
pp. 110-117 ◽  
Author(s):  
Ewelina Łojewska ◽  
Tomasz Kowalczyk ◽  
Szymon Olejniczak ◽  
Tomasz Sakowicz

Author(s):  
Akanksha Gupta ◽  
Abhishek K Tripathi ◽  
Pushpraj S Gupta

Background: Bauhinia variegata Linn. is a native plant of Asia and China. B. variegata is found in tropical regions of the world. It belongs to family Leguminosae. It is used for diarrhea, hemorrhoids, constipation, piles, edema, leprosy, wounds, tumors, etc.  Objective: The objective of the present study was to perform extraction of B. variegata flower and isolation of active constituents from the extract. Materials and Methods: The ethanolic extraction of B. variegata flower was performed using the Soxhlet apparatus. The isolation of active constituents from the extract was performed using chromatographic techniques. In column chromatographic studies, n-hexane- [dichloromethane (DCM)] (2:8) was used as an eluting system and further purified through thin layer chromatography (TLC). Compound A and B were isolated through chromatographic techniques, then the molecular formula and characterization of these compounds were carried out with mass and infrared (IR) spectral analysis. Results and Discussion: The percentage yield of B. variegata ethanolic extract (BVE) was found to be 20.8% w/w. The different fractions were F1 having 12.5 grams with n-hexane, F2 (17.1 grams) with CH2Cl2, F3 (21.2 grams) with EtOAc, and F4 (13.4 grams) with EtOH. Compound A and B were isolated from the solvent fractions of n-hexane-DCM (2:8) and EtOAc-DCM (1:9), respectively. The compound A was characterized as 3-hydroxy-6-methoxy-2-phenyl-4H-chromen-4-one. The compound B was characterized as 3-hydroxy-6-methyl-2-phenyl-4H-chromen-4-one. Conclusion: Thus, B. variegata flowers possess active components that need to identify their biological activities.


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