Strategies For Heterologous Protein Expression In P. Pastoris: Expression Of Human Bone Morphogenetic Protein 4 And 7

Pichia pastoris that is a single-celled eukaryote have been becoming popular since it was developed as heterologous protein expression system, the increasing popularity of this expression system can be attributed to several factors as following: culturing at high cell densities and high grow-rate, easy to genetic manipulation like prokaryote, capable of the posttranslational modifications performed by higher eukaryotic cells. Because Pichia pastoris was developed as expression system in a short time (1993), there are some problems that frustrate this expression system development, the most important of which include: (1) glycosylation from Pichia pastoris differ significantly from those of mammalian cells and humans. Heterologous proteins expressed in Pichia pastoris are added N-linked high-mannose oligosaccharides, hyperglycosylation on recombinant proteins have the potential to trigger immune response and affect folding or function of a foreign protein. (2) attractive levels of expression for any foreign proteins aren't achieved. Although there are few examples of expression of≥1g/L even≥10g/L, expression of many examples reaches ug or mg/L levels. (3) The proteins which propeptide perform a function of intramolecular chaperones are seldom expressed in Pichia pastoris system. The reason that Pichia pastoris can't express the proteins possessed propeptide is that the propeptide isn't cut off from mature peptide because proteases in the secreted pathway of Pichia pastoris differ from those of mammalian. To date several groups have been working on glycosylation engineering in Pichia pastoris with more success, and therefore we plan to research on latter two problems and expect to overcome these problems.Human bone morphogenetic protein4, 7(hBMP4,7) are members of BMPs subfamily belonged to TGF-βsuperfamily, structure of Native hBMP4,7 are homodimer or heterodimer. In this paper, we research influence of rare codon, low RSCU (Relative synonymous codon usage) codon and multiple copies insert on expression of recombinant proteins in Pichia pastoris, and also explore methods to express hBMP4, 7 cDNA in Pichia pastoris.Mature domain of hBMP4/7 were subcloned to pPIC9k vector of Pichia pastoris from pBluescript-hBMP4/7. Recombinant plasmids containing Mature domain of hBMP4/7 were transformed host strain of P. Pastoris. For production of rhBMP4 and rhBMP7 in P. pastoris, flask culture was used. Our results showed rhBMP4 wasn't detected in flask culture and expression levels of rhBMP7 was low, only 5.55mg/L in shake flask.After we analyzed the mature domain of hBMP4/7, we found that RSCU of two Arg codons is zero in the mature domain of hBMP4, and three Arg codons in hBMP7. If do rare codons hamper the expression levels of rhBMP4/7 in P. pastoris? For answering this question, we mutated rare Arg codons (two CGGs in hBMP4, two CGGs and one CGA in hBMP7) to AGA which RSCU is highest by OE-PCR. Mutated mature domains of hBMP4/7 were subcloned to pPIC9K expression vector, recombinant host strains were cultured in shake flask. The results indicated that the expression levels of rhBMP4 were 12mg/L and the expression levels of rhBMP7 reached to 23.59mg/L, expression levels increase by 6 fold compared to native mature domain. Our data shows rare codons influence severely heterologous protein expression in P. pastoris.There are 12 codons which RSCU are between 0.02 and 0.05 in mature domain of hBMP4 except two rare codons. If do the low RSCU codons influence expression of rhBMP4? To potentially solve this problem, we optimized the native mature domain of hBMP4 by means of substituting rare codons and low RSCU codons with more frequently occurring ones and controlled AT nucleotide content of Optimization mature domain between 30%-40%. Optimization sequence was designed by yeast optimization software that was designed according to the characteristics of codon usage in yeast and genetic algorithm by our research group. Optimized mature domain of hBMP4 was transformed into P. pastoris host strain GS115, recombinant strains were expressed in shake flask. In order to assess the actual improvement factor, we compared expression from the optimization sequence to the native sequence inserted at the expression vactor pAO815. Our study showed that the expression levels of optimized mature domain reached to 48mg/L, expression levels increase by 3 fold compared to native mature domain (12mg/L).Some reports showed that recombinant strain that contain multiple integrated copies of an expression cassette often produce larger amounts of foreign protein than do single-copy strains. To examine influence of different copies gene of interest to expression levels of rhBMP7, we constructed recombinant vectors containing 1, 3, 5, 6, 7 and 9 copies mature domain of hBMP7 using pAO815 vector in vitro, and transformed P. pastoris to generate multicopy expression strains. Our results showed that increasing the number of integrated cassettes increased expression levels of rhBMP7, but the relationship between number of integrated cassettes and expression levels wasn't linear. Expression levels of 9 copies were 6-fold higher than that of single copy.To investigate the influence of the fermentation conditions on expression levels and glycosylation of heterologous recombinant protein in Pichia pastoris, various fermentation conditions, e.g., cell density, initial pH, methanol concentration, duration of the induction , temperature and medium volume were studied. To expression levels of rhBMP4, the optimal cell density, initial pH, methanol concentration and frequency of methanol induction in shake flask were OD600=10, pH6.0, 1.5%, and 4-5 (in every 24 h) , respectively. When high cell densities were obtained by fermentation in fermentor, expression levels increased by 5 fold compared to that in shake flask. Fermentation conditions can not significantly influence glycosylation on rhBMP4 and rhBMP7.rhBMP4 and rhBMP7 were purified from the culture supernanant by chromatography, purity of rhBMP4 and rhBMP7 reached to 83% and 90%, respectively. The structures of rhBMP4 and rhBMP7 in P. pastoris were monomer, no dimmer was formed. rhBMP4 and rhBMP7 were glycosylated, we also detected hyperglycosylation on rhBMP4 and rhBMP7. N-linked oligosaccharide side chains produced on rhBMP4 and rhBMP7 expressed in P. pastoris was shown to be hetelogeneous, because a series of diffuse bands at higher molecular mass were present with western blotting analysis.To investigate physiological function of rhBMP4 and rhBMP7, purified rhBMP4 and rhBMP7 were implanted into muscular pouches of the mouse to test the induction ability for new cartilage formation. Histological examination revealed that rhBMP4 and rhBMP7 were able to induce ectopic cartilage formation. Human dermal fibroblast cells were treated with rhBMP4, the functional assays showed that the ALPase activity was increased in a dose- dependent manner. Our data indicated that rhBMP4 and rhBMP7 expressed in P. pastoris possess physiological activity, although the structure of rhBMP4 and rhBMP7 are monomer.Some proteins do not fold into functional proteins in the absence of their propeptides which can function as intramolecular chaperones that facilitate the correct folding of their associated protein. Full sequence of these proteins can not expressed in Pichia pastoris, because their protease sites between propeptide and mature peptide aren't recognized by proteases sorted into secreted pathway of Pichia pastoris. Precursor of hBMP4 and hBMP7 possess propeptide, they can act as good model for study full sequence of heterologous protein expressed in Pichia pastoris. Consensus site R-X-X-R recognized by FURIN protease sorted in Golgi of mammal cells of hBMP4 between propeptide and mature domain was mutated into K-R-E-A site recognized by Kex2 and Ste13 proteases which sort in secreted pathway of Pichia pastoris, and R-X-X-R site of hBMP7 was mutated into K-R cleavage site of Kex2.Another construct containing hBMP4 cDNA sequence was made. All mutated constructs and native constructs of hBMP4 and hBMP7 were expressed in Pichia pastoris, results showed propeptide of hBMP4 was cleaved off from mature peptide after R-X-X-R cleavage site was mutated into K-R-E-A site, but propeptide of hBMP7 wasn't cut off after R-X-X-R cleavage site was mutated into K-R site that only was recognized by Kex2 protease. The data indicates that only K-R cleavage site is not enough for cleaving propeptide from mature peptide and K-R-E-A site can be efficiently recognized and cleaved by Kex2 and Ste13 proteases sorted in secreted pathway of Pichia pastoris. Results also showed that propeptide of hBMP4 is important for its mature peptide correct folding.