Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes

Abstract

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Figure 1

Recursive Directional Ligation. The strategy to double the length of an oligomer in a parent vector involves: (1) digesting the parent vector with both RE1 and RE2 to isolate the ELP insert; (2) digesting the parent vector with only RE1 to linearize the vector, which retains the insert; and (3) dimerizing the two moieties to double the length of the desired gene. The bold areas on the vector represent the endonuclease recognition sequences, which are contained within the sequence that encodes the repetitive polypeptide. The black arrows indicate the location of the cleavage site.

Figure 2

(A) Recursive directional ligation by plasmid reconstruction (PRe-RDL). One round in PRe-RDL involves: (1) purifying the ELP-containing DNA fragment from the parent vector that is digested with AcuI and BglI; and (2) purifying the ELP-containing fragment from the parent vector that is digested with BseRI and BglI; and then (3) ligating the two compatible halves to reconstitute the original vector, and thereby while doubling the length of the insert. (B) Original RDL vector design reported by Meyer , with a representative pentamer sequence. The identity of the capitalized base pairs are specified by the recognition site of the restriction enzyme listed above those nucleotides. Note that the sequence of the restriction endonucleases required for RDL are contained within the DNA sequence that is oligomerized. The vertical arrows indicate the endonuclease restriction sites. (C) PRe-RDL vector, which utilizes the type IIs restriction enzymes, BseRI and AcuI, to eliminate sequence dependence upon the recognition sites. The recognition sequence for BseRI has been designed directly into the Shine-Delgarno ribosomal binding sequence (RBS; underlined), AGGAGGAG, which is required to initiate translation. The BseRI cleavage site (‘CC’ in this vector) is 8-bases downstream of its recognition site. The recognition site for AcuI, CTGAAG, is 14-bases downstream of its degenerate cleavage site, which is ‘GG’ in this vector. The vertical arrows indicate the endonuclease cleavage site on the sense strand. (D) BseRI and AcuI have 2-bp overhangs, which reduces intra-sequence dependence within the repeating unit to a single amino acid. (E) Basic design for a leader sequence to be inserted into a vector restricted with NdeI and BseRI.

Figure 3

PRe-RDL is a modular design that allows for the combination of multiple libraries. This figure demonstrates the stepwise ligation of a leader to an ELP, and then the ligation of a trailer to the Leader-ELP fragment. The ligation order shown here is not essential; leaders were appended to the ELPs before incorporating the trailers, though the order could be reversed if needed.

Figure 4

ELP₁ and ELP₂ libraries produced by PRe-RDL. (A) ELP₁ gene library run on an agarose gel (1%). The left lane represents a size standard ladder, with sizes in base pairs shown on the left. Lanes 2–11 are diagnostic digests of each construct (restricted with XbaI and BamHI, which flank the ELP sequence with 66 bp), with the length shown on the right (in base pairs) and bottom (in pentapeptides). Lanes 2–8 were generated using concatemerization, while 9–11 were created using PRe-RDL. (B) ELP₁ expression library run on an SDS-PAGE gel. The left lane is the Bio-Rad Kaleidoscope^™ Ladder, with lengths in kDa on the left. Lanes 2–6 show the expressed ELPs with the lengths shown on the right (in kDa) and bottom (in pentapeptides). The dimer in lane 4 (ELP₁-60 L₁T₁) is indicative of disulfide bonds formed between the cysteine residues present in T₁. (C) ELP₂ gene library run on an agarose gel. Lanes 2–3 were generated using concatemerization, whereas lanes 4–8 were formed via PRe-RDL. (D) ELP₂ expression library run on an SDS-PAGE gel. To increase expression yields, the ELP₂ library was modified with L₁ (MSKGPG) on the amino terminus.

Figure 5

Thermal properties of the ELP₁ and ELP₂ series. (A) ELP₁ T_t as a function of concentration and chain length. The dashed line represents the predicted T_t for each ELP₁ length based on Meyer and Chilkoti’s model . (B) ELP₂ T_t as a function of concentration and chain length in 1 M NaCl. The ELP₂ T_t was > 80°C at these concentrations. 1 M NaCl was added to depress the T_t to demonstrate the presence of the thermal transition. (C) The ELP T_t as a function of chain length at a constant 25 μM ELP. ELP₁ is in a PBS solution, whereas ELP₂ is in 1 M NaCl in water. The dashed line represents the expected T_t for ELP₁ based on the model developed by Meyer and Chilkoti .