Extensive libraries of gene truncation variants generated by in vitro transposition

Abstract

The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5΄ and 3΄ fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.

Figure 1.

Overview of method to generate random deletions in a target gene. (1) A transposition reaction was performed to obtain random insertions of the transposon into the target gene. (2) 5΄ and 3΄ fragment sub-libraries of the gene were amplified in two separate PCR reactions. In each reaction, one primer was complementary to the 5΄ or 3΄ constant regions of the target gene, and the other to a region on the transposon. (3) The digestion with BsaI created unique overhangs in each library complementary to unique overhangs in the linker DNA (pUC19*-fragment) to favor directional ligation. (4) Libraries were ligated to the linker DNA, which was free of MlyI sites. (5) The product of ligation was treated with MlyI to remove the transposon sequence. (6) Intramolecular blunt-end ligation joined the 5΄ and 3΄ terminal fragments of the gene. (7) This circular library was linearized by PCR with primers complementary to the termini of the parental gene. (8) The final library of deletion variants of the desired size range was isolated by gel electrophoresis.

Figure 2.

5΄ and 3΄ fragment sub-libraries generated from the transposition reaction (TR) by PCR amplification and analyzed on 1% agarose gel. The transposition reaction was performed according to manufacturer instruction and directly used as PCR template. The ‘Inhibited TR’ sample was heated at 75°C for 10 min in the presence of 10 mM ethylenediaminetetraacetic acid prior to the addition of the target gene, incubated at 30°C, and then used for PCR. The transposon alone was used as template for the PCR reaction for the ‘Transposon only’ sample, and ligase 10C parent alone as template for the PCR reaction for the ‘ligase 10C’ sample. M = Marker (100 bp DNA ladder).

Figure 3.

DNA of final deletion library after size-selection by gel extraction shown on a 1% agarose gel. M = Marker (2-Log DNA ladder).

Figure 4.

Theoretically possible and observed counts of deletions at each nucleotide position of the ligase 10C parent gene are shown as empty or open circles, respectively. The number of possible deletions were calculated using Equation (2). Observed counts were obtained from alignments between sequences found in the library and the parental sequence of ligase 10C.

Figure 5.

Deletion lengths observed in the library are shown as percent of theoretically possible deletions of that length (closed circle, trend line shown in grey). The number of possible deletion events for each length was calculated using Equation (3).

Figure 6.

Progress of in vitro selections for ligases using an incubation time during the selection step of 5 min (black bars) or 60 min (grey bars).

Figure 7.

mRNA-displayed proteins after individual steps of selection round 6 analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. (A) Gel for ‘60 min selection’. (B) Gel for ‘5 min selection’. TL: translation; OdT: eluate after purification by oligo-(dT) cellulose chromatography; Flag: eluate from Flag affinity purification; RT: reverse transcription of ³⁵S-Met-labeled protein-RNA fusions to verify efficiency of reverse transcription; RT-³²P: reverse transcription in presence of ³²P-α-dATP to include ³²P-labeling in the cDNA; Dialysis: sample recovered after dialysis into Flag buffer; Flag II: eluate from second Flag affinity purification. The ligation reaction was performed with biotinylated 3΄-OH-substrate in presence of complementary splint oligonucleotide.

Figure 8.

Comparison of DNA from deletion library before round 1 to DNA obtained after 6 rounds of the selections with 5 or 60 min reaction time analyzed by agarose gel electrophoresis (1%). M = Marker (50 bp ladder). It can be clearly seen that the selection process enriched variants similar in length as ligase 10C parent in the ‘5 min selection’, and in addition shorter variants were also enriched in the ‘60 min selection’. This figure is a composite gel picture combining different lanes from the same original agarose gel.

Figure 9.

Sequence alignment of ligase 10C with selected deletion variants from round 6. Ligase variants containing deletions of 13 and 18 amino acids (shown as periods highlighted in gray) were isolated after six rounds of the ‘60 min selection’. The topmost sequence is the original ligase 10C (31). The second sequence shows ligase 10C parent which comprises an additional N-terminal Flag tag for purification purposes during the selection. Dashes symbolize amino acids that are identical to the original ligase 10C sequence. Numbering of residues corresponds to numbering used in the first publication of ligase 10C (31).

Figure 10.

Ligation progress over time catalyzed by the three deletion variants (dashed or dotted gray lines) compared to original ligase 10C activity (solid black line). Rates of ligation were 0.387 ± 0.094 h⁻¹, 0.304 ± 0.120 h⁻¹ and 0.412 ± 0.059 h⁻¹ for Del 13 ΔFlag, Del 18 ΔFlag and Del 18 I22T ΔFlag, respectively, while k_obs of ligase 10C was 0.340 ± 0.020 h⁻¹. For each variant, the error bars represent the standard error from at least 3 biological replicates for which 2–4 technical replicates were performed.