High-level Expression Of Barnacle Adhensive Protein Mrcp20K In Pichia Pastoris

It is one of the current research hotspots to develop new,stable,high-efficiency adhesives which can be used in water environments from the adhesive substances secreted by the attached organisms.Among them,because the adhesion process does not depend on the dopa molecular system which needs complex amino acid modification,the barnacle gum has strong adhesion and anti-scouring properties,thus is an ideal material for the development of underwater high-performance biomimetic adhesive.However,in nature,barnacle secretes little gum proteins and easily polymerize.Consequently,it is difficult to obtain barnacle gum in large scale,and a large number of samples are consumed in its production,resulting in extremely high cost.Therefore,how to prepare barnacle gum in large quantity has become an urgent scientific problem to be solved.In vitro,gene recombination technology may provide a feasible technical scheme.Among the existing expression hosts,Pichia pastoris is recognized as an excellent one for high-efficiency expression of heterologous proteins,and has successfully expressed a variety of heterologous proteins.Therefore,in this study,a viscous protein Mrcp20 K from Megabalanus rosa was taken as the research object,via the combined strategies,such as fusion expression with Rhizopus oryzae lipase,increasing gene dosage,co-expression with helper-proteins,and high-density fermentation,the expression level of Mrcp20 K protein was increased to gram level,which is the highest one of adhensive protein expression as reported so far.This research provides a strong technical support for the engineering application of barnacle viscous proteins.The main contents and results of this study are summarized below.1.The gene Mrcp20 k of viscous protein from red giant barnacle was integrated into the pPICZ?A vector,and then was transformed into the P.pastoris X-33 in a single copy.The supernatant of the shaking flask fermentation was measured by the Coomassie brilliant blue method,and the total crude protein was 62 ± 3.5 mg/L,suggesting that barnacle adhensive protein can be successfully expressed in yeast.2.Using the “bio-brick” method,the pPICZ?A-Mrcp20 k vectors with 1,2,3,and 4copies of the gene were successfully constructed and respectively transformed into the P.pastoris X-33.After shaking flask fermentation,the supernatant was tested,and the highest crude protein of pPICZ?A-4Mrcp20 k could reach 172 ± 7.6 mg/L.The clone X-33/pPICZ?A-4Mrcp20 k 4# with the best protein expression was selected to optimize shaking flask fermentation conditions.When the optimized conditions were methanol induction time 96 h,induction temperature 25?,and initial inoculation volume 3%,the amount of methanol 1.5% and the initial pH of the medium 7.0,the average total crude protein of the supernatant was enhanced to 182 ± 4.52 mg/L.3.To further improve the expression of Mrcp20 k in P.pastoris,the gene of Mrcp20 k was fused with that of Rhizopus oryzae lipase to form the fusion gene rol-Mrcp20 k so that the highly effective secretion system of Rhizopus oryzae lipase could be employed.The fused gene proROL-Mrcp20 k was then integrated into the pAO?N-ROL vector,and by using the “bio-brick” method,the pAO?N-Mrcp20 k vectors with 1,2,3,and 4 copies of the fusion gene were successfully constructed and respectively transformed P.pastoris GS115 host.The supernatant was detected by the BBC method after shaking flask fermentation,and the total crude protein of 4 copies of pAO?N-4Mrcp20 k was the highest,attaining 410 ± 10.0 mg/L,which is 1.95 times of a single copy of pAO?N-Mrcp20k(210± 5.5 mg/L).4.After co-expressing Bmh2,Sso2,Ssa4 and VHb with(rol-4Mrcp20 k pAO?NproROL-4Mrcp20k-Bmh2-Sso2-Ssa4-VHb)in the Pichia pastoris GS115,the average total crude protein in the supernatant of shaking flask fermentation further increased to 499 ±10.9 mg/L.Then,the clone of GS115/pAO?N-proROL-4Mrcp20k-Bmh2-Sso2-Ssa4-VHb8# with the best protein expression was selected to optimize shake flask fermentation conditions.Under the optimal conditions: methanol induction time 96 h,induction temperature 25 ?,the initial inoculation amount 3%,methanol loading 1.5%,and the initial pH 7.0,the average total crude protein of the supernatant was up to 621 ± 9.0 mg/L.Furthermore,the strain GS115/pAO?N-proROL-4Mrcp20k-Bmh2-Sso2-Ssa4-VHb was also subjected to high-density fermentation in a 50-L fermentor,the average total crude protein in the supernatant was measured to 7.6 g /L.In addition,a prelim characterization experiment was carried out on Mrcp20 K and recombinant ROL-Mrcp20 K,and the results of XPS analysis showed that the two proteins have a certain degree of adhesion.