4 Fungal immunomodulatory proteins Based on the amino acid sequence,LZ-8 has very high homology gene is expressed in E coli BL21 cells,Ultimately,the yield is with FIP-fve,and it suggests that LZ-8 and FIP-fve should have increased up to 70 mg/L,which is more than twice compared very similar crystal structures.An et al expressed reLZ-8 using with other researchers (Bai et al.,2006).For another thing,this prokaryotic expression system and studied on its structure by X- gene can also be cloned into another expression vector pET-28a ray crystallography.The results showed that the form of reLZ-8, and expressed in Ecoli BL21 cells.Meanwhile,the recombinant which appeared as dimmer,was the same with natural LZ-8, protein is account for 36.25%of total protein (Li et al.,2009).In connected by non-covalent bond (An et al.,2010).This research addition,FIP-gsi gene can also be cloned into vector pET-30a and laid the foundation for the further study of the function and then expressed in E.coli BL21 cells.The recombined protein is application of FIPs represented by LZ-8.Studied on the secondary mainly insoluble.And the yield is account for 36.25%of total structure of reLZ-8 expressed in Pichia pastoris GS115,the helix protein (Liet al,2011).The FIP-fiegene is cloned from the genome and B forms are calculated by the circular dichroism assay and the DNA of F veltipes and expressed in E coli BL21.The recombinant result showed that the a-helix:B-fold:B-turn was in proportion of expression vectors pET-28(+)-FIP-fve are reconstructed and then 1:4:1 in reLZ-8 (proportion of 2:7:1 in natural protein)and 1:3:1 transformed into E coli BL21.And the yield of the recombinant in reFIP-fve (proportion of 3:6:1 in natural protein).Meanwhile, FIP-fve is about 30 mg/L (Xu et al.,2009). the sugar content test showed that:reLZ-8 contained 1.8% The E.coli M15 cell is another main host cell.1Z-8 (FIP-glu) carbohydrates,reFIP-fve contained 1.2%carbohydrates (the that was cloned into the pQE-30 expression vector can also be natural LZ-8 contains 1.3%carbohydrate,and natural FIP-fve is expressed in EcoliM15 cells(Liet al.,2009).FIP-gsiis transformed a pure protein)(Lin et al.,2009).The results showed that these into pQE-30 expression vector and expressed in E coli M15 cells. two recombinant proteins contained low levels ofa-helix,and were Using pQE-30 expression vector expressed in E.coli M15 cells, glycosylated in various degrees.In addition,the hydrophobic loop LZ-8(FIP-glu)and FIP-gsiare mostly soluble recombinant protein, region near the C-terminus of LZ-8 contained sequences of Val- they accounted for 19.84%and 25%of total soluble protein, Asp-Pro-Asp-Thr-Asn-Asn-Asp-Phe,which is similar to Ca2. respectively (Li,2010).In some cases,E coli TGI cells are also binding site sequences.However,the mechanism of their biological used as FIPs host cells.Lin etal reported that recombinant FIP-gts activity is not clear (Murasugi et al.,1991). was expressed as glutathione S-transferase fusion protein in Ecoli Studies on high-resolution protein structure of FIPs are rare. with a yield of 20 mg/L (Lin et al.,1997). Wu et al cloned FIP-gmi gene from G.microsporim and expressed FIP-fve cDNA is amplified by polymerase chain reaction(PCR), FIP-gmi in P pastoris.They got its 2.0 Astructure(Figure 4).FIP- then ligated into the expression vector pGEX-2T,and expressed gmi appears in the form of tetramer instead of dimmer,which is fusion protein of glutathione S-transferase(GST)and FIP-fve in formed by rich non-covalent and hydrophobic interactions though E coli.The GST-FIP-fve fusion protein is soluble,and the yield of the interface of o-helix in the N-terminal.The conformation and recombinant FIP-fve is about 5 mg/L after induced (Ko et al., arrangement of loops at the neighbor of residues 64 and 105 are 1997).Besides,Yeh et al have expressed 1Z-8(FIP-gl)gene in different from those corresponding regions of FIP-fve.Unlike LZ- Bacillus subtilis and Lactococcus lactis.Similarly,they synthesize 8,FIP-gmi shows more thermal sensitivity,and it would lose its recombinant 1Z-8 by overlapping extension PCR,using the biological activity even at room temperature (Wu et al.,2007). preferred codons for both strains (Yeh et al.,2008). EXPRESSION SYSTEMS OF FIPs Eukaryotic Expression System Gene expression is the process in which information in a gene Yeast is a kind of lower eukaryotes,but it is a good expression is translated to functional protein product.So far,the gene system for eukaryotic genes.It can overcome the disadvantages encoding FIPs has only been effectively expressed in prokaryotes on the lack of protein translational processing and modification and eukaryotes yeast.. in E coli.Therefore,yeast expression system receives more and more attention and is nowadays commonly used.So far,studies Prokaryotic Expression System on FIPs expression in yeast mainly use methanol-based yeast P pastoris.The advantages are that it has highly efficient promoter Among the existing expression systems,the first one used for Pand the expressed protein is not secreted,which makes study is prokaryotic expression system,which is currently the most purification easier.It also has a comparatively high yield and low developed system.Precious researches proved that the expression level of glycosylation (Song et al.,2003). of FIPs occurred more frequently in prokaryotic host cells,such Presently,the strain selected in FIPs expression system is P pastoris as E.coli. GS115 strain.The expression levels of recombinant proteins are As expression host bacterial strain,Ecoli have always played an 191.2mg/L (reLZ-8)(Lin et al.,2009a)and 158mg/L (reFIP-fve) important role in prokaryotic expression of FIPs.BL21 competent (Lin,2009),respectively.1Z8 gene is ligated into two different cells can express many kinds of FIPs.Taking advantage of the vectors,and the protein expression levels are different.They are preferred codons of E coli,Huang et al replaces 8 species of rare 191.2mg/L (pPIC9 vector)(Lin,2009)and 270mg/L(pPIC9K codons in 1Z-8 gene with preferred codons in E coli cells(Huang vector),respectively (Xue et al,2008).Nevertheless,after being et al.,2008).Ligased with pET28b vector,the optimized LZ-8 cultivated in a 100-1 fermentation tank,the reLZ-8 protein yield4 Fungal immunomodulatory proteins Based on the amino acid sequence, LZ-8 has very high homology with FIP-fve, and it suggests that LZ-8 and FIP-fve should have very similar crystal structures. An et al. expressed reLZ-8 using prokaryotic expression system and studied on its structure by X￾ray crystallography. The results showed that the form of reLZ-8, which appeared as dimmer, was the same with natural LZ-8, connected by non-covalent bond (An et al., 2010). This research laid the foundation for the further study of the function and application of FIPs represented by LZ-8. Studied on the secondary structure of reLZ-8 expressed in Pichia pastoris GS115, the helix and β forms are calculated by the circular dichroism assay and the result showed that the α-helix:β-fold:β-turn was in proportion of 1:4:1 in reLZ-8 (proportion of 2:7:1 in natural protein) and 1:3:1 in reFIP-fve (proportion of 3:6:1 in natural protein). Meanwhile, the sugar content test showed that: reLZ-8 contained 1.8% carbohydrates, reFIP-fve contained 1.2% carbohydrates (the natural LZ-8 contains 1.3% carbohydrate, and natural FIP-fve is a pure protein) (Lin et al., 2009). The results showed that these two recombinant proteins contained low levels of α-helix, and were glycosylated in various degrees. In addition, the hydrophobic loop region near the C-terminus of LZ-8 contained sequences of Val￾Asp-Pro-Asp-Thr-Asn-Asn-Asp-Phe, which is similar to Ca2+ binding site sequences. However, the mechanism of their biological activity is not clear (Murasugi et al., 1991). Studies on high-resolution protein structure of FIPs are rare. Wu et al. cloned FIP-gmi gene from G. microsporum and expressed FIP-gmi in P. pastoris. They got its 2.0 Å structure (Figure 4). FIP￾gmi appears in the form of tetramer instead of dimmer, which is formed by rich non-covalent and hydrophobic interactions though the interface of α-helix in the N-terminal. The conformation and arrangement of loops at the neighbor of residues 64 and 105 are different from those corresponding regions of FIP-fve. Unlike LZ- 8, FIP-gmi shows more thermal sensitivity, and it would lose its biological activity even at room temperature (Wu et al., 2007). EXPRESSION SYSTEMS OF FIPs Gene expression is the process in which information in a gene is translated to functional protein product. So far, the gene encoding FIPs has only been effectively expressed in prokaryotes and eukaryotes yeast.. Prokaryotic Expression System Among the existing expression systems, the first one used for study is prokaryotic expression system, which is currently the most developed system. Precious researches proved that the expression of FIPs occurred more frequently in prokaryotic host cells, such as E. coli. As expression host bacterial strain, E. coli have always played an important role in prokaryotic expression of FIPs. BL21 competent cells can express many kinds of FIPs. Taking advantage of the preferred codons of E. coli, Huang et al. replaces 8 species of rare codons in LZ-8 gene with preferred codons in E. coli cells (Huang et al., 2008). Ligased with pET28b vector, the optimized LZ-8 gene is expressed in E. coli BL21 cells, Ultimately, the yield is increased up to 70 mg/L, which is more than twice compared with other researchers (Bai et al., 2006). For another thing, this gene can also be cloned into another expression vector pET-28a and expressed in E. coli BL21 cells. Meanwhile, the recombinant protein is account for 36.25% of total protein (Li et al., 2009). In addition, FIP-gsi gene can also be cloned into vector pET-30a and then expressed in E. coli BL21 cells. The recombined protein is mainly insoluble. And the yield is account for 36.25% of total protein (Li et al., 2011). The FIP-fve gene is cloned from the genome DNA of F. velutipes and expressed in E.coli BL21. The recombinant expression vectors pET-28(+)-FIP-fve are reconstructed and then transformed into E. coli BL21. And the yield of the recombinant FIP-fve is about 30 mg/L (Xu et al., 2009). The E. coli M15 cell is another main host cell. LZ-8 (FIP-glu) that was cloned into the pQE-30 expression vector can also be expressed in E. coli M15 cells (Li et al., 2009). FIP-gsi is transformed into pQE-30 expression vector and expressed in E. coli M15 cells. Using pQE-30 expression vector expressed in E. coli M15 cells, LZ-8 (FIP-glu) and FIP-gsi are mostly soluble recombinant protein, they accounted for 19.84% and 25% of total soluble protein, respectively (Li, 2010). In some cases, E. coli TG1 cells are also used as FIPs host cells. Lin et al. reported that recombinant FIP-gts was expressed as glutathione S-transferase fusion protein in E. coli with a yield of 20 mg/L (Lin et al., 1997). FIP-fve cDNA is amplified by polymerase chain reaction (PCR), then ligated into the expression vector pGEX-2T, and expressed fusion protein of glutathione S-transferase (GST) and FIP-fve in E. coli. The GST-FIP-fve fusion protein is soluble, and the yield of recombinant FIP-fve is about 5 mg/L after induced (Ko et al., 1997). Besides, Yeh et al. have expressed LZ-8 (FIP-glu) gene in Bacillus subtilis and Lactococcus lactis. Similarly, they synthesize recombinant LZ-8 by overlapping extension PCR, using the preferred codons for both strains (Yeh et al., 2008). Eukaryotic Expression System Yeast is a kind of lower eukaryotes, but it is a good expression system for eukaryotic genes. It can overcome the disadvantages on the lack of protein translational processing and modification in E. coli. Therefore, yeast expression system receives more and more attention and is nowadays commonly used. So far, studies on FIPs expression in yeast mainly use methanol-based yeast P. pastoris. The advantages are that it has highly efficient promoter PAOX, and the expressed protein is not secreted, which makes purification easier. It also has a comparatively high yield and low level of glycosylation (Song et al., 2003). Presently, the strain selected in FIPs expression system is P. pastoris GS115 strain. The expression levels of recombinant proteins are 191.2mg/L (reLZ-8) (Lin et al., 2009a) and 158mg/L (reFIP-fve) (Lin, 2009), respectively. LZ-8 gene is ligated into two different vectors, and the protein expression levels are different. They are 191.2mg/L (pPIC9 vector) (Lin, 2009) and 270mg/L(pPIC9K vector), respectively (Xue et al., 2008). Nevertheless, after being cultivated in a 100-l fermentation tank, the reLZ-8 protein yield