Department of Neural and Behavioral Sciences, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America.
PLoS One. 2010 Nov 30;5(11):e15056. doi: 10.1371/journal.pone.0015056.
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 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.
色素上皮衍生因子（PEDF）因其神经营养和抗血管生成功能而广为人知。由于异源蛋白产量低，PEDF 在动物模型中的功效研究受到限制。在这里，我们重新设计了人 PEDF 基因，使其优先匹配大肠杆菌的密码子频率，而不改变氨基酸序列。经过从头合成，密码子优化的 PEDF（coPEDF）和 wtPEDF 基因被克隆到 pET32a 中，其中包含 5'硫氧还蛋白序列（Trx）和重组 Trx-coPEDF 或 Trx-wtPEDF 融合构建体，在天然和两个 tRNA 增强的大肠杆菌宿主 - BL21-CodonPlus（DE3）-RIL 和 BL21-CodonPlus（DE3）-RP 中表达，分别携带额外的 tRNAarg、ile、leu 和 tRNAarg、pro 基因。使用 Ni-NTA 金属亲和层析分离 Trx-PEDF 融合蛋白，并用因子 Xα 切割后纯化 PEDF。通过 Western blot、MALDI-TOF 和 UV/CD 光谱分析确认蛋白质纯度和身份。与天然宿主相比，合成基因的表达约增加 3.4 倍（212.7 mg/g；62.1 mg/g 湿细胞），纯化产率约增加 4 倍（41.1 mg/g；11.3 mg/g 湿细胞）。与天然宿主相比，添加稀有 tRNA 基因的宿主中两种基因的表达均略有增加，但 coPEDF 的表达在天然和密码子偏倚调整的大肠杆菌菌株中均比 wtPEDF 高约 3 倍。两个融合基因的 Trx 位点的 ΔGs 在-3 到+50 为-3.9 kcal/mol。功能上，coPEDF 与 wtPEDF 一样有效，可降低氧化应激、促进神经突生长和阻断内皮管形成。这些发现表明，虽然稀有 tRNA 扩增和 mRNA 折叠能显著促进蛋白质表达，但在这种情况下，有利的密码子用法有利于大肠杆菌中生物活性 PEDF 的翻译效率。该策略无疑将加速研究，以验证 PEDF 在体内的治疗效用。
