scholarly journals A technique to increase protein yield in a rabbit reticulocyte lysate translation system

BioTechniques ◽  
2014 ◽  
Vol 56 (1) ◽  
Author(s):  
Maria Anastasina ◽  
Ilya Terenin ◽  
Sarah J Butcher ◽  
Denis E Kainov
Keyword(s):  
Protein Yield ◽  
Rabbit Reticulocyte Lysate ◽  
Translation System ◽  
Reticulocyte Lysate

Role of the ribosome in sequence-specific regulation of membrane targeting and translocation of P-glycoprotein signal-anchor transmembrane segments

2000 ◽  
Vol 113 (14) ◽  
pp. 2545-2555
Author(s):  
J.T. Zhang ◽  
E. Han ◽  
Y. Liu
Keyword(s):  
Membrane Proteins ◽  
Wheat Germ ◽  
Rabbit Reticulocyte Lysate ◽  
Translation System ◽  
Membrane Targeting ◽  
Combined System ◽  
P Glycoprotein ◽  
Reticulocyte Lysate ◽  
Signal Anchor ◽  
Specific Regulation

It is thought that the topology of a polytopic protein is generated by sequential translocation and membrane integration of independent signal-anchor and stop-transfer sequences. Two well-characterized cell-free systems (rabbit reticulocyte lysate and wheat germ extract) have been widely used to study the biogenesis of secretory and membrane proteins, but different results have been observed with proteins expressed in these two different systems. For example, different topologies of P-glycoprotein (Pgp) were observed in the two systems and the cause was thought to be the source of ribosomes. To understand how the ribosome is involved in dictating membrane translocation and orientation of polytopic proteins, individual signal-anchor sequences of Pgp were dissected and examined for their membrane targeting and translocation in a combined system of wheat germ ribosomes (WGR) and rabbit reticulocyte lysate (RRL). Addition of wheat germ ribosomes to the rabbit reticulocyte lysate translation system can enhance, reduce, or have no effect on the membrane targeting and translocation of individual Pgp signal-anchor sequences, and these effects appear to be determined by the amino acid residues flanking each signal-anchor. Ribosomes regulate the membrane targeting and translocation of Pgp signal-anchors in a polytopic form differently from the same signal-anchors in isolation. Furthermore, we demonstrated that ribosomes regulate the membrane targeting and translocation of each signal-anchor cotranslationally and that this activity of ribosomes is associated with the 60S subunit. Based on this and previous studies, we propose a mechanism by which ribosomes dynamically dictate the membrane targeting and translocation of nascent polytopic membrane proteins.

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