Study Of An Indirect P.pastoris Surface Display Method Based On The Im7/CL7 Ultra-High-Affinity System And Its Application In Enzyme Immobilization

Direct and indirect surface display systems based on yeast cells are broadly studied and utilized in research of life science and industry.A highly efficient indirect P.pastoris surface display method based on the Im7/CL7 Ultra-High-Affinity system and its application in immobilization of human Arginase ? were reported here.The SED1 protein from S.cerevisiae was used as P.pastoris surface anchoring motif.SED1 fusion proteins with one Im7 protein,or tow and three Im7 proteins linked with glycine-serine peptide linker were displayed on surfaces of P.pastoris GS115 cells,respectively.The molecular numbers of CL7-sf GFP proteins indirectly displayed on various types of P.pastoris cells were assayed through fluorometric assay.The enzymatic features of free CL7 fused human Arginase ? were studied.The activities of free and Im7 protein displaying P.pastoris cell immobilized CL7 fused human Arginase ? were assayed.The P.pastoris strain directly displaying human Arginase ? was constructed and compared with that indirectly displayed on P.pastoris surface based on the Im7/CL7 ultral-high-affinty system.The enzymatic features of P.pastoris cell surface directly displayed human Arginase ? were studied and the enzymatic activity was assayed.The main research contents and results of this report are showed in the following text.1.The plasmids p ET23a-CL7-sf GFP,p ET23a-CL7-m Cherry and p ET23a-CL7-hu Arg I for the expression of CL7 fused sf GFP,m Cherry and human Arginase ? were constructed through PCR amplification and T5 exonuclease-dependent assembly.The fusion proteins CL7-sf GFP,CL7-m Cherry and CL7-hu Arg I were expressed in E.coli BL21(DE3)cells and purified with Ni²⁺-affinity chromatography.The fluorescent intensity calibration curve of CL7-sf GFP fusion proteins in p H 7.6 TBS buffer was prepared(y=0.0982x-0.1421;y:fluorescent intensity of the CL7-sf GFP fusion protein;x:the concentration of the CL7-sf GFP fusion protein,?g/m L;R²=0.9922).2.The P.pastoris surface display vector p PICZ?A-HA-SED1 was constructed through PCR amplification and T5 exonuclease-dependent assembly first and then the Im7,2×Im7 and3×Im7 P.pastoris surface display plasmids p PICZ?A-HA-Im7-SED1,p PICZ?A-HA-2×Im7-SED1 and p PICZ?A-HA-3×Im7-SED1 were constructed as described above;These plasmids were linearized by restriction endonuclease Pme I and electroporated into GS115 competent cells;the strains GS115/p PICZ?A-HA-SED1,GS115/p PICZ?A-HA-Im7-SED1,GS115/p PICZ?A-HA-2×Im7-SED1 and GS115/p PICZ?A-HA-3×Im7-SED1 were constructed and confirmed through yeast colony PCR first and then these yeast cells displaying SED1 fusion proteins were analyzed with flow cytometry,fluorescence microscopy,blue light transmitter and Western Blot;the highly efficient P.pastoris cell surface display of SED1 fusion proteins and the specific binding of Im7 proteins with CL7-sf GFP and CL7-m Cherry fusion proteins were confirmed;the molecular numbers of CL7-sf GFP fusion proteins indirectly displayed on yeast cells were assayed through fluorometric assay after induction with methonol for 24,48,72 and 96 hours.The results indictating that these yeast cells were saturated by Im7 proteins after induction with methonol for 72 hours.The molecular numbers of CL7-sf GFP fusion proteins indirectly displayed on the yeast GS115/p PICZ?A-HA-Im7-SED1,GS115/p PICZ?A-HA-2×Im7-SED1 and GS115/p PICZ?A-HA-3×Im7-SED1 cells induced with methonol for96 hours was about 2.830×10⁶,2.946×10⁶ and 4.274×10⁶,respectively.3.The concentration calibration curve of L-ornithine was prepared using the Chinard colorimetric assay;the enzymatic features of free CL7-hu Arg I fusion proteins were studied;the activities of free and GS115/p PICZ?A-HA-3×Im7-SED1 cell surface immobilized CL7-hu Arg I fusion proteins were assayed;the optimal p H,temperature and Mn²⁺concentration were 9.0,60?and 2 m M,respectively;at optimal reaction conditions,the activity of free CL7-hu Arg I fusion proteins was about 909.0 U/mg,the activity of CL7-hu Arg I fusion proteins immobilized on GS115/p PICZ?A-HA-3×Im7-SED1 cells was about 217.8 U/mg;the activities of wet and dry P.pastoris cells were 1804 U/g and 6910 U/g,respectively;the residual activity of CL7-hu Arg I fusion proteins was 24%after immobilized on yeast cells.4.The human Arginase ? P.pastoris display vector p PICZ?A-HA-hu Arg I-SED1 was constructed through PCR amplification and T5 exonuclease-dependent assembly.The plasmid was linearized by restriction endonuclease Pme I and electroporated into GS115 competent cells;the yeast strain GS115/p PICZ?A-HA-hu Arg I-SED1 was constructed and confirmed through yeast colony PCR first and then analyzed with flow cytometry,fluorescence microscopy and Western Blot;the highly efficient P.pastoris cell surface display of human Arginase ? was confirmed;the enzymatic features of the GS115/p PICZ?A-HA-hu Arg I-SED1cell surface displayed human Arginase ? were studied;the optimal p H,temperature and Mn²⁺concentration of the GS115/p PICZ?A-HA-hu Arg I-SED1 cell surface displayed human Arginase ? were 9.0,60?and 2 m M,respectively;the activity of GS115/p PICZ?A-HA-hu Arg I-SED1 cells under the optimal conditions was assayed and the enzyme activities of 1g of wet and dry GS115/p PICZ?A-HA-hu Arg I-SED1 cells were 1398 U and 5356 U,respectively.