Heterogonous Expression Of An Unknown Expansin-like Protein From Trichoderma Reesei

Expansin is the unique protein from plants been found recently, which has the ability to induce heat-inactivated cell wall to elongation. However, there are some proteins, having the same ability, have been found in some other species which can infect or digest cellulose as well. Those proteins are called expansin-like protein. Although researchers have been trying to express expansin and expansin-like protein in heterogeneous expression system since the family of expansin has been reported, there is no report showed that it can be done successfully. Nowadays, scientists focus on looking for novel expansin-like protein from microbe and recombinant expression in heterogonous expression system in order to substitute expansin to apply to industry.This article concentrates on the research on the gene of an unknown expansin-like protein from fungus Trichoderma reesei. The protein has been confirmed as the first reported expansin-like protein from Trichoderma reesei by database analysis. We cloned the gene to Escherichia coli express system and Pichia pastoris for heterogenous expression. The result shows that the recombinant expression of the expansin-like protein showed that no hydrolytic enzymes activity or expansin-like activity could be detected in E.coli, though the gene could express few soluble recombinant proteins in E. coli. We synthetize reverse mutated gene, since there are two different amino acids between the sequence of recombinant expressed protein from amplification and the two original amino acid sequences, YNEAGIWTPKNVF and YNAEAWYNIVHTGWV, from Edmam degradation. The reverse mutated gene could express soluble recombinant protein in E. coli as well. Still, no hydrolytic enzymes activity or expansin-like activity could be detected. We can infer that the recombinant protein showing no activity in E.coli is not due to the difference of amino acids. The reason may be the inaccurance folding of polypeptide chain or the glycosylation of the protein.The expansin-like protein gene can not be heterogenous expressed in P.pastoris. No recombinant protein can be detected in the culture and the cell lysation. The culture and the cell lysation show neither hydrolytic enzymes activity nor expansin-like activity. Allowing for the gene came from fungus and had lots of rare codes, we attempt to optimize codes of the expansin-like protein gene sequence for P. pastoris. However, the optimized gene could not express in P. pastoris express system neither. The unsuccessful expression in P.pastoris may not be caused by the rare codes but the failing transcription of mRNA. So, through making use of strong promoter pCBHI, we constructed express vehicle for trailing it in T.reesei, a more complex fungus express system, hoping for getting recombined expansin-like protein in order to study the characteristic of this novel expansin-like protein further.