Gene cloning, splicing overlap extension PCR (SOE-PCR), and cell growth for protein overexpression

The gene encoding TON-LPL (NCBI Reference Sequence {"type":"entrez-protein","attrs":{"text":"ACJ16398","term_id":"212009016"}}ACJ16398) from Thermococcus onnurineus NA1 was PCR amplified using Prime STAR HS DNA polymerase premix (DSS Takara Bio India Pvt. Ltd., New Delhi, India).The forward primer-5′-ATTTTAGGTACCGTGAGGGTTTACAAAGCCAAGTTCGGTGAGC-3 and reverse primer-5′-TTAATAAAGCTTATTCTCTTTTCTTCCTCCCGCGTG-3′ (restriction sites are written in italics) were used in the PCR. The PCR amplified product were digested by KpnI and HindIII. Subsequently, it was ligated into the predigested pQE30 vector. The ligation products were transformed into XL1 Blue, and positive colonies were selected for protein overexpression studies. Protein was expressed by growing the cells at 37 °C for 24 h with constant shaking at 200 rpm in 1 L terrific broth autoinduction medium with trace elements (AIM-Terrific Broth, Hi-Media Laboratories Pvt. Ltd., Mumbai, India) or in LB broth (induced by 1 mM IPTG).

The chimera gene encoding rc-TGL was constructed by splicing overlap extension PCR (SOE-PCR) reaction using two gene fragments: one fragment was amplified from Thermomyces lanuginosus lipase gene (TLIP), and another fragment was from TON-LPL gene. The forward and reverse primers used are listed in Additional file 3: 3.3. The SOE-PCR amplified product was digested by NdeI and XhoI and ligated into pET-23a vector having a 6×-His tag at its C-terminus. The ligation products were transformed into XL1 Blue and the positive clones were confirmed by restriction digestion analysis of the isolated plasmid (Additional file 3: 3.2). The pET-23a plasmid carrying rc-TGL gene was transformed into BL21 (DE3) cells for protein expression. Protein was expressed by growing the BL21 (DE3) cells at 37 °C for 24 h in 1 L terrific broth autoinduction medium with trace elements (Hi-Media Laboratories Pvt. Ltd., Mumbai, India).