Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.

BACKGROUND: The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. RESULTS: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae alpha-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of approximately 3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to approximately 1.5 g of 99% pure recombinant human insulin per liter of culture broth. CONCLUSIONS: A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

Improving human interferon-beta production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene.

Human beta-interferon is used extensively as a therapeutic agent in a wide variety of diseases, ranging from multiple sclerosis to viral infections. At present, the most common source of interferon-beta is derived from CHO (Chinese-hamster ovary) cells. Interestingly, however, the IFNB gene is characterized by a lack of intronic sequences and therefore does not undergo splicing during its expression pathway. As nuclear processing of pre-mRNA molecules has often been demonstrated to improve production yields of recombinant molecules, we have inserted a heterologous intronic sequence at different positions within the IFNB gene and analysed its effects on protein production. The results obtained in the present study show that the position of intron insertion has profound effects on the expression levels of the IFNB gene and on the nuclear/cytoplasm distribution levels of its mRNA as determined by FISH (fluorescent in situ hybridization) analysis of stably transfected clones. In conclusion, our results provide additional evidence that insertion of intronic sequences may be used to improve protein expression efficiency also in molecules that do not normally undergo any splicing process.

A Simplified and Efficient Process for Insulin Production in Pichia pastoris.

A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.

Mix-infections with different genotypes of HCV and with HCV plus other hepatitis viruses in patients with hepatitis C in China.

AIM: Clinical therapy and prognosis in HCV infections are not good, and mix-infections with different HCV genotypes or quasispecies and mix-infections with HCV plus other hepatitis viruses are important concerns worldwide. The present report describes the sequence diversity and genotying of the 5'NCR of HCV isolates from hepatitis patients mix-infected with different HCV genotypes or variants, and the conditions of mix-infections with HCV plus other hepatitis viruses, providing important diagnostic and prognostic information for more effective treatment of HCV infections. METHODS: The 5' non-coding region (5'NCR) of HCV was isolated from the patients sera and sequenced, and sequence variability and genotypes of HCV were defined by nucleotide sequence alignment and phylogenetic analysis, and the patients mix-infected with HCV plus other hepatitis viruses were analyzed. The conditions and clinical significance of mix-infections with HCV plus other hepatitis viruses were further studied. RESULTS: Twenty-four out of 43 patients with chronic hepatitis C were defined as mix-infected with different genotypes of HCV. Among these 24 patients, 9 were mix-infected with genotype 1 and 3, 7 with different variants of genotype 1, 2 with different variants of genotype 2, 6 with different variants of genotype 3. No patients were found mix-infected with genotype 1 and 2 or with genotype 2 and 3. The clinical virological analysis of 60 patients mix-infected with HCV plus other hepatitis viruses showed that 45.0 % of the patients were mix-infected with HCV plus HAV, 61.7 % with HCV plus HBV, 6.7 % with HCV plus HDV/HBV, 8.4 % with HCV plus HEV, 3.3 % with HCV plus HGV. Infections with HCV plus other hepatitis viruses may exacerbate the pathological lesion of the liver. CONCLUSION: The findings in the present study imply that mix-infections with different HCV genotypes and mix-infections with HCV plus other hepatitis viruses were relatively high in Yunnan, China, providing important diagnostic and prognostic information for more effective treatment of HCV infections.

Dual role of dextran sulfate 5000 Da as anti-apoptotic and pro-autophagy agent.

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.

InTRONs in biotech.

Eukaryotic gene expression relies on several complex molecular machineries that act in a highly coordinated fashion. These machineries govern all the different steps of mRNA maturation, from gene transcription and pre-mRNA processing in the nucleus to the export of the mRNA to the cytoplasm and its translation. In particular, the pre-mRNA splicing process consists in the joining together of sequences (known as "exons") that have to be differentiated from their intervening sequences commonly referred to as "introns." The complex required to perform this process is a very dynamic macromolecular ribonucleoprotein assembly that functions as an enzyme, and is called the "spliceosome." Because of its flexibility, the splicing process represents one of the main mechanisms of qualitative and quantitative regulation of gene expression in eukaryotic genomes. This flexibility is mainly due to the possibility of alternatively recognizing the various exons that are present in a pre-mRNA molecule and therefore enabling the possibility of obtaining multiple transcripts from the same gene. However, regulation of gene expression by the spliceosome is also achieved through its ability to influence many other gene expression steps that include transcription, mRNA export, mRNA stability, and even protein translation. Therefore, from a biotechnological point of view the splicing process can be exploited to improve production strategies and processes of molecules of interest. In this work, we have aimed to provide an overview on how biotechnology applications may benefit from the introduction of introns within a sequence of interest.

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability.

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.

TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing.

TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.

Lack of evidence of sexual transmission of hepatitis C among monogamous couples: results of a 10-year prospective follow-up study.

The risk of sexual transmission of hepatitis C virus (HCV) infection was evaluated among 895 monogamous heterosexual partners of HCV chronically infected individuals in a long-term prospective study, which provided a follow-up period of 8,060 person-years. Seven hundred and seventy-six (86.7%) spouses were followed for 10 yr, corresponding to 7,760 person-years of observation. One hundred and nineteen (13.3%) spouses (69 whose infected partners cleared the virus following treatment and 50 who ended their relationship or were lost at follow-up) contributed an additional 300 person-years. All couples denied practicing anal intercourse or sex during menstruation, as well as condom use. The average weekly rate of sexual intercourse was 1.8. Three HCV infections were observed during follow-up corresponding to an incidence rate of 0.37 per 1,000 person-years. However, the infecting HCV genotype in one spouse (2a) was different from that of the partner (1b), clearly excluding sexual transmission. The remaining two couples had concordant genotypes, but sequence analysis of the NS5b region of the HCV genome, coupled with phylogenetic analysis showed that the corresponding partners carried different viral isolates, again excluding the possibility of intraspousal transmission of HCV. Our data indicate that the risk of sexual transmission of HCV within heterosexual monogamous couples is extremely low or even null. No general recommendations for condom use seem required for individuals in monogamous partnerships with HCV-infected partners.