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Article: Codon preference optimization increases heterologous PEDF expression

TitleCodon preference optimization increases heterologous PEDF expression
Authors
Issue Date2010
PublisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.action
Citation
Plos One, 2010, v. 5 n. 11 How to Cite?
AbstractPigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNA arg,ile,leu and tRNA arg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was,3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ~4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ~3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo. © 2010 Gvritishvili, et al.
Persistent Identifierhttp://hdl.handle.net/10722/169862
ISSN
2015 Impact Factor: 3.057
2015 SCImago Journal Rankings: 1.395
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorGvritishvili, AGen_HK
dc.contributor.authorLeung, KWen_HK
dc.contributor.authorTombranTink, Jen_HK
dc.date.accessioned2012-10-25T04:57:09Z-
dc.date.available2012-10-25T04:57:09Z-
dc.date.issued2010en_HK
dc.identifier.citationPlos One, 2010, v. 5 n. 11en_HK
dc.identifier.issn1932-6203en_HK
dc.identifier.urihttp://hdl.handle.net/10722/169862-
dc.description.abstractPigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNA arg,ile,leu and tRNA arg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was,3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ~4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ~3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo. © 2010 Gvritishvili, et al.en_HK
dc.languageengen_US
dc.publisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.actionen_HK
dc.relation.ispartofPLoS ONEen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.titleCodon preference optimization increases heterologous PEDF expressionen_HK
dc.typeArticleen_HK
dc.identifier.emailLeung, KW: kwleung1@hku.hken_HK
dc.identifier.authorityLeung, KW=rp01674en_HK
dc.description.naturepublished_or_final_versionen_US
dc.identifier.doi10.1371/journal.pone.0015056en_HK
dc.identifier.pmid21152082-
dc.identifier.scopuseid_2-s2.0-78649775828en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-78649775828&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume5en_HK
dc.identifier.issue11en_HK
dc.identifier.isiWOS:000284755100056-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridGvritishvili, AG=15834526600en_HK
dc.identifier.scopusauthoridLeung, KW=13106059300en_HK
dc.identifier.scopusauthoridTombranTink, J=7003724753en_HK

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