Modular polypeptide libraries and methods of making and using same
一种模块化、文库的技术，应用在制备合成模块化多肽文库和编码合成模块化多肽文库的核酸,细胞中与合成模块化多肽相关的所选表型,并且其中所述刺激物是抗领域，能够解决限制新合成蛋白开发速率、过于繁琐和昂贵、繁重等问题
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Embodiment 1
[0436] Example 1: Construction and screening of its regulatory domain modular library
[0437] To prepare nucleic acid fragments of barcoded coding modules containing the necessary elements for library construction, nucleic acids encoding polypeptide modules (i.e. co-regulatory domains (i.e. co-stimulatory or co-repressive)) were subcloned into DNA suitable for sequencing and IIS type restriction endonuclease digested cloning vector. After subcloning, the vector contains: module coding sequence, optimal Gly / Ser linker sequence flanking module coding sequence, module specific barcode sequence, 3' cloning homology arms flanking module specific barcode sequence, A BamHI restriction enzyme site between the module coding sequence and the module-specific barcode sequence and a type IIS restriction endonuclease site on the 5' end of the module coding sequence and the 3' end of the module-specific barcode sequence ( figure 1 ). The subcloned vector inserts were sequenced to confir...
Embodiment 2
[0450] Example 2: Confirmation of comprehensive assembly of a 61×61 two-dimensional library
[0451] The 61 variable CAR modules (Table 2, provided in Figure 25 Middle) Assembly into barcoded nucleic acid libraries encoding two-dimensional (2D) synthetic modular CAR polypeptides. The library was deep-sequenced by a sequencing-by-synthesis (SBS) approach using the MiSeq system (Illumina Inc., Hayward, CA) and the read count for each assembled library member was determined ( Figure 26 ).
[0452]Out of a total of 3,721 possible library members (ie, CAR variants), all possible library members were detected by sequencing with a maximum occurrence of 1216 counts and a minimum occurrence of 2 counts. The mean number of counts in the library was 333 with a median of 311 counts and a standard deviation of 140.90% of the library members were represented as having counts within 2 times the median and 98% of the library members had 3 times the median Count within .
[0453] Thus,...
Embodiment 3
[0454] Example 3: Library Partial Normalization
[0455] A method was developed to enhance the distribution of clones in combinatorial libraries using library fraction normalization.
[0456] In any combinatorial assembly reaction, some parts (such as modules) are more efficiently incorporated into the assembly product than others. As a result, assembly products (eg, protein variants) that contain relatively inefficiently integrated parts are underrepresented or absent in the final combinatorial library. For similar reasons, other assembly products, such as those protein variants that contain efficiently integrated parts, are overabundant and, therefore, oversampled in all downstream assays.
[0457] To address this issue, a method for improved library assembly was developed. In the method, an initial assembly reaction is performed in which each part (DNA insert) is present in the same volume. For this purpose, generate a master mix containing 1 µL of each fraction. Afte...
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