A Single Pot Cloning Method for Protein Hybrid Generation

Abstract

Engineering proteins for diverse applications by using evolutionary strategies is currently an active and intensively explored area of research. Molecular cloning techniques started developing drastically ever since the first restriction endonuclease enzyme was discovered. Here we report a simple single pot method to generate a library of protein hybrids/chimeras from unrelated parents. This method allows unbiased facile DNA shuffling based recombination between multiple parents from unrelated sources by introducing a type IIB restriction endonuclease, BsaXI site at multiple distinct crossover sites. To assess this methodology, the immunoglobulin binding B1 domain of protein G and another parent which is a computationally designed protein top7 were recombined at four sites (both computationally and experimentally) to yield 2 5 (32) possible hybrid proteins along with wild type parent proteins. Tandem colony multiplex PCR based screening was followed to screen the above generated hybrids and, the resulting library of hybrid proteins were analyzed through oligonucleotide sequencing, gel electrophoresis and protein expression levels under different conditions. These hybrids were further analyzed for their thermodynamic stability by studying observed changes in their intrinsic fluorescence properties upon chemical and thermal denaturation, to generate a range of free energy of unfolding values (4.2 to 5.6 kcal/mol). Hence we have successfully demonstrated a novel single pot cloning method, that experimentally yielded five protein hybrids possessing varying range of stable folded secondary structures. newline