Chen Dequan, Langford Marlyn P, Duggan Chris, Madden Benjamin J, Edwards Albert O
Institute for Retina Research, Presbyterian Hospital, 8210 Walnut Hill Lane, Dallas, TX 75231, USA.
Protein Expr Purif. 2007 Aug;54(2):275-82. doi: 10.1016/j.pep.2007.03.017. Epub 2007 Apr 3.
LOC387715 is a hypothetical gene located on human chromosome 10q26.13 that is associated with the development of age-related macular degeneration (AMD). The native open reading frame (ORF) of LOC387715 cDNA - LOC387715(ORF), contains a large number of Escherichia coli (E. coli) rare codons (RC) including 5.6% and 15.0% Group-I and IIa translational problem causative (TPC) RCs, respectively, which forms 3 and 4 simple E. coli rare codon clusters (RCC) where RCs are spaced by 1 and 2 respective non-TPC codons and one complex E. coli RCC where RCs and RCCs are spaced by <5 non-TPC codons. We modified the entire 35 E. coli RCs (6, 16 and 13 Group I, IIa and IIb RCs, respectively) present in LOC387715(ORF) with their optimal or sub-optimal synonymous degenerate codons, and the resulted LOC387715(ORF)m was free from Shine-Dalgarno-like sequence (SDLS) and ribosome binding site complementary sequence (RBSCS). SDS-PAGE and Western blotting analysis demonstrated that LOC387715(ORF)m was capable of highly expressing the recombinant protein rLOC387715 in E. coli. Mass spectrometry analysis indicated that the bacterial expressed rLOC387715 contained the correct and expected amino acid (aa) sequence without aa misincorporation, aa missing or frame-shift. The results suggest that high and authentic expression of LOC387715 recombinant protein in E. coli was achieved by the synonymous modification of its native ORF cDNA sequence for all the 3 groups of bacterial RCs and the simultaneous elimination of SDLS and RBSCS sequences.
LOC387715是一个位于人类10号染色体q26.13上的假设基因，与年龄相关性黄斑变性（AMD）的发生发展有关。LOC387715 cDNA的天然开放阅读框（ORF）——LOC387715(ORF)，包含大量大肠杆菌（E. coli）稀有密码子（RC），其中I组和IIa组导致翻译问题（TPC）的稀有密码子分别占5.6%和15.0%，形成了3个和4个简单的大肠杆菌稀有密码子簇（RCC），其中稀有密码子分别被1个和2个非TPC密码子隔开，还有一个复杂的大肠杆菌RCC，其中稀有密码子和稀有密码子簇被少于5个非TPC密码子隔开。我们用其最佳或次优同义简并密码子修饰了LOC387715(ORF)中存在的全部35个大肠杆菌稀有密码子（分别为6个、16个和13个I组、IIa组和IIb组稀有密码子），得到的LOC387715(ORF)m没有类似Shine-Dalgarno序列（SDLS）和核糖体结合位点互补序列（RBSCS）。SDS-PAGE和蛋白质免疫印迹分析表明，LOC387715(ORF)m能够在大肠杆菌中高效表达重组蛋白rLOC387715。质谱分析表明，细菌表达的rLOC387715包含正确且预期的氨基酸（aa）序列，没有氨基酸错掺入、氨基酸缺失或移码。结果表明，通过对其天然ORF cDNA序列的所有3组细菌稀有密码子进行同义修饰，并同时消除SDLS和RBSCS序列，实现了LOC387715重组蛋白在大肠杆菌中的高效和真实表达。
