Functional proteins from a random-sequence library

Abstract

Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3' end of its encoding mRNA, to sample a large number of distinct random sequences. Starting from a library of 6 x 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries.

Figure 1

In vitro selection and amplification of mRNA-displayed proteins. The DNA library encodes proteins with 80 contiguous random amino acids. Transcription, splinted ligation to a 3′-puromycin oligonucleotide, translation, high-salt incubation, purification and reverse transcription (RT) yielded 6 × 10¹² independent mRNA-displayed proteins. This library was incubated with an ATP-agarose affinity matrix. Unbound material was washed away with selection buffer at 4 °C, and bound material was collected by incubation with the same buffer containing 5 mM ATP at 4 °C. Eluted fractions were combined, amplified by PCR, and used as the input for the next round of in vitro selection and amplification.

Figure 2

Proportion of the mRNA-displayed protein library bound to immobilized ATP and subsequently eluted with free ATP, as a function of selection round. The inputs into rounds 10–12 were subjected to mutagenic PCR amplification with an average mutagenesis rate of 3.7% at the amino-acid level per amplification.

Figure 3

Sequences of selected ATP-binding proteins. a, Consensus sequences of the four selected protein families at round 8 (A, B, C, D), before mutagenesis. Flanking constant region residues are indicated by dashes. These protein sequences are unrelated to each other or to any known biological protein. b, The consensus sequence of the single remaining protein family at round 18 (18predom). Four predominating substitutions (>39/56 clones) and sixteen other selectively enriched (>4/56 clones) substitutions are also indicated (18select). Clone 18–19 had the lowest K_d for ATP (100 nM) of those assayed. c, Deletion analysis of clone 18–19 using DNA-tagged proteins. Purified constructs were incubated with ATP-agarose, washed and eluted with free ATP. The fraction present in the elution phase is shown, normalized with respect to the full-length DNA-tagged protein. Invariant residues are indicated by dashes. d, Deletion analysis of clone 18–19 using MBP fusion proteins. Successive deletion constructs of clone 18–19 were generated as MBP fusion proteins, then cleaved from the MBP with thrombin. Apparent K_ds were determined by displacement equilibrium filtration. Invariant residues are indicated by dashes.

Figure 4

Dissociation constants of protein–ATP (and ATP analogue) complexes as determined by displacement equilibrium filtration for protein 18–19 as an MBP fusion protein.