Linked Life Data

21152082

Source:http://linkedlifedata.com/resource/pubmed/id/21152082

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2010-12-14
pubmed:abstractText
Pigment 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 tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor X?. 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. ?Gs 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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1932-6203
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
5
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
e15056
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Codon preference optimization increases heterologous PEDF expression.
pubmed:affiliation
Department of Neural and Behavioral Sciences, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't