Methods of Altering Expression of Polypeptides
一种表达系统、重组多肽的技术，应用在仪器、DNA制备、生物系统等方向，能够解决多肽表达生理化学参数和过程未得到充分理解等问题
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example 1
[0269] Example 1: mRNA signatures that control protein expression levels in E. coli
[0270] Assessed the expression of 6,348 protein-coding genes from diverse phylogenetic sources ( Figure 15 ). The protein-coding gene was transcribed from the bacteriophage T7 promoter in pET21, a 5.4 kb pBR322-derived plastid with an ampicillin resistance marker (Acton, T.B. et al. (2005) Methods Enzymol 394, 210-243). This dataset provides a broad sampling of the codon space due to variations in codon usage frequency in different organisms. Bacteriophage polymerases were used to drive transcription to minimize possible confounding effects of coupling translation and transcription due to native E. coli RNA polymerase (Iost, I. et al. (1995) Embo j 14, 3252-3261; Iost, I. et al. (1992) J Bacteriol 174, 619-622). Protein expression was induced overnight at 18°C in E. coli strain BL21λ(DE3) (Acton, T.B. et al. (2005) Methods Enzymol 394, 210-243). E. coli strain BL21λ(DE3) has a single c...
example 2
[0298] Example 2: Model M for Predicting the Probability of High Protein Expression Levels for RNA Sequences
[0299] The codon repetition rate is defined as r=:, where d i is to codon c i The distance to the next occurrence. For example, "AAA.CGT.CCG.CGT.AAA" r = mean (1 / 4, 1 / 2, 0, 0, 0) = 3 / 20. Binary multiple logistic regression is highly expressed log odds of the explanatory variable x i The linear model, θ=log[E_5 / E_0]=A+∑ i beta i x i . The predicted high expression probability is: The number of degrees of freedom for codon variables is due to the constraint 1 = Σf c and less than the number of codons. In the multivariate logistic analysis in Figure 11, ATG is removed such that the slope β ATG = 0, its contribution is absorbed into the constant A. R Statistical Program [R Core Team (2013). R is a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http: / / www.R-project.org / ] to calculate model parameter...
example 3
[0304] Example 3: Method for constructing synonymous sequences
[0305] Synonymous sequences were designed in two ways and then tested experimentally. In the 6AA method, the codons for six amino acids were changed to those specified in Table 1. Although no explicit free energy optimization was performed with the 6AA method, the average free energy density was also more favorable in the genes tested. In the 31C-FO approach, the free energy of the head-end+pET21 expression vector was optimized to be as high as possible (i.e., with the weakest mRNA secondary structure), using only codons from the subset listed in Table 1 below, And the free energy of the tail is optimized to be close to -10kcal / mol for the 48-mer nucleotide window. In 31C-FD, a subset of codons is used to deoptimize the free energy to be as low as possible (with the strongest mRNA secondary structure).
[0306] Table 1:
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