Abstract
Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2 of 40 min at 70°C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.
Highlights
The extracellular recombinant enzyme production simplifies recovery and downstream processing of the target protein
Codon analysis of Ba-Gt-amy revealed the feasibility of its expression in P. pastoris (Supplementary Table S2)
Theoretically the copy number of the expression cassettes integrated into the P. pastoris determines the expression level of heterologous protein, because increasing the gene dosage will increase the specific transcript and the increased transcript level leads to an increase in specific translation
Summary
The extracellular recombinant enzyme production simplifies recovery and downstream processing of the target protein. P. pastoris expression system has provided a platform for commercialization of a wide range of protein products such as enzymes, antigens and antibodies. Numerous examples can be cited from the literature indicating that an increase in gene copy number can significantly increase productivity provided that secretion level should not reach saturation, which suggests that there is a linear correlation between copy number and expression of heterologous proteins (Romanos et al, 1998; Vassileva et al, 2001; Aw and Polizzi, 2013)
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