Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants.
The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin-like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)(n) that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin-10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5-40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80-160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C-terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N-terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification.
Conley
Andrew J
AJ
Department of Biology, University of Western Ontario, London, Canada.
Joensuu
Jussi J
JJ
Jevnikar
Anthony M
AM
Menassa
Rima
R
Brandle
Jim E
JE
D020816:Amino Acid Motifs; D015870:Gene Expression; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D014026:Nicotiana
D011993:Recombinant Fusion Proteins