The High Expression Of Recombinant Trypsinogen In Pichia Pastoris

Trypsin is a serine protease,found in the digestive system of many vertebrates,where it hydrolyses proteins.It is considered an endopeptidase,which cleaves peptide chains mainly at the carboxyl side of the amino acidslysine or arginine,except when either is followed by proline.Trypsin is one of the most specific proteases,and serves as vital tools in determination of structure of proteins and polypeptides.Currently,trypsin is produced from animal-source and non-animal source.The traditional sources of trypsin have been bovine or porcine pancreatic tissues,neither of which is acceptable under the current regulatory definitions as primarily and secondarily animal-origin free.The non-animal source trypsin is made through genetic engineering recombinant technology.In this thesis,to address demands of high-quality protein production,recombinant trypsinogen was prepared by transforming porcine trypsinogen gene into host P.pastoris and created a recombinant enzyme unrivaled in performance and purity.These results demonstrated that the non-animal sourced trypsin appeared to function equivalently to serine proteases derived from animal tissues and may exhibit other practical advantages for small-scale and production-scale cell culture applications.It is anticipated that these non-animal trypsin substitutes will reduce the risk of introducing adventitious virus to adherent cultures and will serve as a superior or alternative to porcine trypsin for regulated applications.Firstly,recombinant expression plasmid?Referring to European patent EP1399568B1?was built by molecular cloning technique.The recombinant expression plasmid was transferred into the host of GS115 by electricity transformation to express target protein inductively.A high-yield restructuring engineering bacteria was obtained by plenty of screening.The cell strain was named P.pastoris GS115/pPIC9K-Bd P4 WB54.Secondly,the morphological,biochemical and genetic characteristics of the engineering bacteria were studied.And the main cell bank and working cell bank of the strain were also established.A comprehensive study of the cell's generation stability was carried out to determine whether the cell bank was stable and could express the target protein continuously.These results showed that the recombinant pancreatic proteinase engineered bacterium had the typical morphological and biochemical characteristics of pichia yeast,and the target gene was accurately integrated into the host cell genome.The engineered bacteria were stable after 30generations,which indicated that the recombinant gene was stable and the target protein was continuously expressed.Thirdly,the fermentation process of the obtained engineering strain was optimized in 50 L test scale,including inoculation time,inoculation amount,culture medium,induction induced bacteria concentration,pH,temperature,methanol feeding rate,organic nitrogen source adding quantity,urea feeding rate and fermentation period,etc.The results showed that the expression level of 7.5 g/L of the recombinant trypsinogen was obtained under the optimized conditions.The specific optimal conditions were as follows:the best inoculation time:?21²4h?*(OD₆₀₀:19³1),the best inoculation proportion:5%?v/v?,the best BGJZ medium:?85%H₃PO₄ 13 mL/L?glycerol40 g/L?CaSO₄·2H₂O 0.5 g/L?MgSO₄·7H₂O 7 g/L?KOH 2g/L?PTM1solution 4.35 m L/L?urea 6 g/L and GPE defoamer 2 mL/L?,the best induced cell concentration:OD₆₀₀:170±5,the best induced pH:4.0,the best temperature:25?,the best Methanol feeding rate:7.0 g/L/h,the best Organic nitrogen source YEB medium?1%yeast extract powder and 2%soy peptone?feeding rate:2.0 g/L/h,the best urea feeding amount:2.0%,the best fermentation period:110h.Finally,the optimal fermentation process of recombinant bacteria and optimized optimum fermentation process were enlarged to 5000 L.The results showed that the engineering bacteria and the optimized fermentation could amplify to product scale smoothly.The various technological parameters were consistent with that of 50 L scale.In addition,the bacteria concentration(OD₆₀₀)and trypsinogen yield can also arrive at the test level.The recombinant expression level reached to 7.5 g/L.Above all,this experiment has successfully built engineering bacteria used to produce trypsinogen.Meanwhile,the fermentation conditions of the engineering bacteria were also studied.Eventually,the high expression of trypsinogen was obtained.This thesis could provide the practical guidance for the industrialized production of trypsinogen.