scholarly journals Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland

1979 ◽  
Vol 181 (3) ◽  
pp. 737-756 ◽  
Author(s):  
R K Craig ◽  
A P Boulton ◽  
O S Harrison ◽  
D Parker ◽  
P N Campbell
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Complexity Analysis ◽  
Free Protein ◽  
Mrna Species ◽  
Run Off ◽  
Membrane Bound ◽  
Cell Free Protein Synthesis

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87–93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

scholarly journals Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland

1984 ◽  
Vol 222 (2) ◽  
pp. 501-510 ◽  
Author(s):  
A P Boulton ◽  
J C Pascall ◽  
R K Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Secretory Pathway ◽  
Short Pulse ◽  
Early Event ◽  
Post Translational Modification ◽  
Electrophoretic Mobilities ◽  
Membrane Bound

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.

scholarly journals Cell-free protein synthesis and in situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion

RSC Advances ◽  
2020 ◽  
Vol 10 (66) ◽  
pp. 40588-40596
Author(s):  
Tony Köhler ◽  
Thomas Heida ◽  
Sandra Hoefgen ◽  
Niclas Weigel ◽  
Vito Valiante ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genetic Information ◽  
In Situ Immobilization ◽  
Bottom Up ◽  
Free Protein ◽  
Malonyl Coa ◽  
Cell Free Protein Synthesis

We describe a bottom-up approach towards functional enzymes utilizing microgels as carriers for genetic information that enable cell-free protein synthesis, in situ immobilization, and utilization of functional deGFP-MatB.

scholarly journals Multivariate statistical data analysis of cell‐free protein synthesis toward monitoring and control

AIChE Journal ◽  
2021 ◽  
Author(s):  
Carlos A. Duran‐Villalobos ◽  
Olotu Ogonah ◽  
Beatrice Melinek ◽  
Daniel G. Bracewell ◽  
Trevor Hallam ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Data Analysis ◽  
Statistical Data ◽  
Monitoring And Control ◽  
Statistical Data Analysis ◽  
Multivariate Statistical ◽  
Free Protein ◽  
And Control ◽  
Cell Free Protein Synthesis

scholarly journals Cell-free riboswitches

2021 ◽  
Author(s):  
Takeshi Tabuchi ◽  
Yohei Yokobayashi
Keyword(s):  
Protein Synthesis ◽  
The State ◽  
Future Directions ◽  
Free Protein ◽  
Current Perspective ◽  
Gene Switches ◽  
A Current ◽  
Cell Free Protein Synthesis

Synthetic riboswitches can be used as chemical gene switches in cell-free protein synthesis systems. We provide a current perspective on the state of cell-free riboswitch technologies and their future directions.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

scholarly journals De novo expression and antibacterial potential of four lactoferricin peptides in cell-free protein synthesis system

2021 ◽  
Vol 29 ◽  
pp. e00583
Author(s):  
Nawal Abd El-Baky ◽  
Maie Ahmed Elkhawaga ◽  
Eman Shawky Abdelkhalek ◽  
Mona Mohammed Sharaf ◽  
Elrashdy Mustafa Redwan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
De Novo ◽  
Synthesis System ◽  
Free Protein ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis ◽  
Antibacterial Potential
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