Thermal sensitive and highly hydrophobic elastin like polypeptide (ELP) tends to undergo inverse transition cycling (ITC) which can be used for non chromatographic purification. The present study reports non-chromatographic purification of industrially important α-amylase tagged with ELP and compared with IMAC (Immobilized metal affinity chromatography) purified α-amylase. amyL-Gene encoding α-amylase from Bacillus licheniformis was cloned and expressed in E. coli BL21. The expressed protein with His-tag was purified through IMAC using Ni–NTA matrix. Three ELP genes encoding repeats of pentapeptide (Val-Pro-Gly-Val-Gly)n with variable length (V = 20, 21, 22) were synthesized through PCR using overlapping primers. To generate ELP tagged α-amylase, amyL was placed at N-terminal of ELPs and transformed to E. coli BL21 for expression. After ITC, ELP22 at 30 °C showed maximum yield. α-Amylase purification through ITC and IMAC showed 2.9 and 1.72-fold purification, respectively. Furthermore, physical parameters of ELP tagged α-amylase have shown improvement with working temperature and thermal stability in comparison to His-tag α-amylase. The kcat/Km for ELP tag and His-tag α-amylase was found to be 61.4 and 23.7 mg−1 min−1, respectively, which shows that ELP-tag increased the enzyme efficiency. In conclusion, the ELP-tag purification strategy can be applied to industrially relevant enzyme for purification by the non-chromatography method.
α-Amylase Elastin like polypeptide Inverse transition cycling Transition temperature Hydrophobic peptide
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The authors wish to thank the Department of Biotechnology (DBT) (Project No BT/PR9727/GBD/27/505/2013), New Delhi for financial support.
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Conflict of interest
The authors do not have any conflict of interest to declare.
Nilsson J, Stahl S, Lundeberg J, Uhlén M, Nygren PA (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 11:1–16CrossRefPubMedGoogle Scholar
Urry DW (1988) Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics. J Protein Chem 7:1–34CrossRefPubMedGoogle Scholar
Trabbic-Carlson K, Meyer DE, Liu L, Piervincenzi R, Nath LaBean T, Chilkoti A (2004) Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: A role for surface hydrophobicity? Protein Eng Des Sel 17:57–66. doi:10.1093/protein/gzh006CrossRefPubMedGoogle Scholar
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–429CrossRefGoogle Scholar
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
Urry DW, Luan CH, Parker TM, Gowda DC, Prasad KU, Reid MC, Safavy A (1999) Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity. J Am Chem Soc 113:4346–4348. doi:10.1021/ja00011a057CrossRefGoogle Scholar
Krishnan T, Chandra AK (1983) Purification and characterization of α-amylase from Bacillus licheniformis CUMC305. Appl Environ Microbiol 46:430–437PubMedPubMedCentralGoogle Scholar
Moscaelli P, Boraldi F, Bochicchio B, Pepe A, Salvi AM, Quaglino D (2014) Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3. Matrix Biol 36:15–27CrossRefGoogle Scholar
Urry DW, Trapane TL, Prasad KU (1985) Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers 24:2345–2356. doi:10.1002/bip.360241212CrossRefPubMedGoogle Scholar