In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

Abstract

BackgroundProtein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized.Principal FindingsWe report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents.ConclusionThe shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

“…3b). This kinetic constant is comparable to the transsplicing kinetic constant of 6.6 ± 1.3 Â 10 À5 (s À1 ) reported for SspDnaE although the comparison between trans-splicing kinetics could not be directly compared because of the differences in the extein sequences [18,38]. The molecular mass of 15 899.7 Da was obtained for the ligated product by MALDI-TOF mass-spectrometry ( Supplementary Fig.…”

“…The plasmid (pHYDuet93) for the expression of an N-terminal His-tagged B1 domain of IgG binding protein G (H 6 -GB1) fused with NpuInt NDC6 was constructed from pSKDuet16 containing the gene of the single chain variant NpuDnaE intein and two GB1s as exteins by replacing the codon of residue 132 with a stop codon by two oligonucleotides: #HK254: 5 0 -CACTCAAAAATTAAGCTTTA-GCTTCTAATTGTTTC and #HK255: 5 0 -GAAGCTAAAGCTTAATTTTT-GAGTGCAAAATTATG [16,18]. This plasmid contains RSF origin and a T7 promoter that is inducible by isopropyl b-D-1-thiogalactopyranoside (IPTG) [7].…”

“…The NMR solution structure and the backbone dynamics of NpuDnaE intein were analyzed in order to rationally design new split inteins that could constitute a shorter and functional C-or Nintein for protein ligation by protein trans-splicing [18,20]. Because there was no three-dimensional structure of SspDnaE intein available when the project started [37], we previously shifted the split site of SspDnaE systematically toward the C-terminus and discovered that only the C-terminal 16 residues were required for the functional C-intein of SspDnaE [18].…”

“…Because there was no three-dimensional structure of SspDnaE intein available when the project started [37], we previously shifted the split site of SspDnaE systematically toward the C-terminus and discovered that only the C-terminal 16 residues were required for the functional C-intein of SspDnaE [18]. This split site was also applied to NpuDnaE intein and demonstrated that the C-terminal 15 residues of NpuDnaE intein were sufficient for efficient protein transsplicing [18]. This split site is located in the loop between b12 and b13 of the NpuDnaE intein structure (Fig.…”

“…We have been interested in identifying shorter functional C-inteins that still retain robust protein trans-splicing activity. A functional C-intein consisting of 15 residues (NpuInt C15 ) was previously identified in our group by shortening the C-intein systematically [18,19]. The systematic approach was applied because no three-dimensional structure of DnaE intein was available when the project was started [18].…”