Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

BACKGROUND: Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. RESULTS: We compared titer and quality of several Nanobodies(®) produced in wild type and Mut(S) strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in Mut(S) strains contained a product variant with a Δ-16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-L-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. CONCLUSION: We showed that misincorporation of methoxine occurs particularly in P. pastoris Mut(S) strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen.

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B-cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1-DE and 2-DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1-D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen ß chain. 2-D analysis revealed the subunits of fibrinogen, especially the ß and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa-antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA-specific epitopes, should be considered as a trigger in tolerance break.