Cloning And Prokaryotic Expression Of Transferrin From Yak

In order to gain the Tf gene of Tianzhu white yak, the first strand was synthesized based on the total RNA extracted from the liver tissue, the Tf coding sequence was amplified by RT-PCR. And it was analyzed by bioinformatics, then the fragments were linked into the vector pET30a(+) and transformed into Escherichia coil BL21(DE3) strain. The recombinant protein was expressed and-purified after identified by PCR、restriction enzyme digestion and nucleotide sequencing. For further study of structure and function of Transferrin, the fermentation has been applied by using the 3L fermentation tank; All of this offered the practice and theoretical guidance to large-scale industrial production. The results of this study are as follows:1. Complete CDS of Tf with 2,115 bp in Tianzhu white was cloned by RT-PCR (GenBank accession number:KP720624), which encode 704 amino acids (AA). Tf protein contains a signal peptide with 19 AA long in the N terminal and two highly conserved homologous domains, which was 42% homology in AA sequence between them. The molecular weight and PI of Tf protein is 75.78 KDa and 6.63, respectively.2. Sequence analysis showed that four base variations were found in the nucleotide sequence of Tf, including two transitions of non-synonymous mutation in A/G in 57th and in T/C in 750th, two transitions of mis-sense mutation in A/T in 482nd and in T/C in 511st, leading Lin 161st and Lin 171st to G and F.3. The homology of AA sequence in Tf protein between Tianzhu white yak and wild yak, bovine, buffalo, Tibetan antelope, sheep, wild boar, human, mouse, chicken and fish was 100%,99%,96%,94%,93%,75%,69%,64%,51% and 39%, respectively.4. In premise of not changing the amino acid coding sequences, we fully consider and adjust the appropriate GC content, the secondary structure of protein translation initiation blocking sequence and optimize the rare codon of target protein in prokaryotic host bacteria, then we remove the signal peptide amino acid sequence encoded by Tf itself. GC content of Tf gene sequence after optimization is 50%, the hairpin structure of 2 small hairpin structure near the initiation codon mRNA and 2 medium had been optimizated,253 nucleotides were changed. The objective gene was obtained by total gene synthesis, construction of recombinant pGH-DT1219-Tf.5. The prokaryotic expression vector of Tf pET-30a-Tf was constructed. With E.coil BL21 (DE3) as the host strain and IPTG as the inducer, we selected the optimum expression conditions (18 C, IPTG final concentration of 0.5mM,16h), and identified the molecular weight by SDS-PAGE is 76 kDa. After high density fermentation laboratory 3L fermentation,7.20mg recombinant protein was obtained with 0.80mg/mL concentration and more than 70% purity.