Discovery new method create libraries ‘circularly permuted’ proteins

Discovery of new method to create libraries of ‘circularly permuted’ proteins

3:31 AM, 20th February 2012
Discovery of new method to create libraries of ‘circularly permuted’ proteins
Manan Mehta, Undergraduate Student, Rice University; Jonathan Silberg, Bioengineer, Rice University; Shirley Liu, Research Technician, Rice University.

HOUSTON, US: Manan Mehta, Undergraduate Student, Rice University discovered a method to create libraries of ‘circularly permuted’ proteins at the suggestion of his mentor, Jonathan Silberg, Bioengineer, Rice University. In a process Manan calls ‘permutation using transposase engineering’ (PERMUTE), he created variations of a protein, in this case, adenylate kinase, that have their amino acids rearranged. The library of mutant proteins was then mined for variants that retained the ability to function by introducing them into Escherichia coli bacteria.

Libraries are particularly useful for scientists who study the rules governing the adaptation of proteins during molecular evolution; they also are useful for designing biosensors and molecular switches with novel functions for synthetic biology. “Existing methods for rearranging the bits of information in a protein are slow and arduous to use. In addition, they are non-ideal because they simultaneously create multiple types of mutations, the desired rearrangements and undesired deletions of important amino acids,” said Silberg.

But with our method, you only generate mutants with rearranged sequences, and you don’t need to be an expert in biomolecular engineering. All you need is the DNA that encodes your gene of interest, the artificial minitransposon we engineered and an enzyme. Mix them all together and you get a library of every possible variant. “It’s a way of making, with great control, all of the diversity of this type of mutation that could exist in a protein,” explained Silberg.

Proteins, strings of amino acids that regulate biological processes, are created in cells. They have a beginning ‘N’ terminus and an end the ‘C’ terminus. “The ‘N’ comes off the ribosome first and builds up from there. Then the proteins fold, like a ball of string. We connect the two ends of the string and break it elsewhere,” said Silberg. “There’s already an enzyme that does most of the work for you. It’s called a transposase, and it inserts the transposon. The hard part is making the transposon itself, which is really just a big piece of DNA,” said Mehta.

Since beginning the project last spring, Mehta has spent half his time in Silberg’s lab modeling his minitransposon at the computer and half implementing his ideas on the bench. The long process of creating the minitransposon involved building his designed DNA and debugging it so that it functioned as intended.

“We ended up with 15 unique variants of permuted adenylate kinase after sequencing 220 or so, about half of which were active and half not. It’s quite an ordeal. Synopsis is a new way of mutating protein in a controlled way. I want to make it simple. It’s clear when you make the libraries that there’s going to be useful diversity in there, but we’re not smart enough to know which mutations to make, so we still need to make the libraries. Making a better library makes it more likely that we’ll find what we want,” said Silberg.

© Rice University News

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