Kyuboem Han

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate.

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.

Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration

An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder. The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E. coli cellular protein as r-fGH. The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment. The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration. More than 47% of the r-fGH was recovered from the E. coli cell lysates and the purity of recovered r-fGH was 98%. The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 micrograms r-fGH g-1 fish body weight, resulted in significant increases both in weight and length. These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.

Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

ABSTRACT Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.

Influence of reducing agents on the secretion rate of recombinant erythropoietin from CHO cells

The specific secretion rate of recombinant erythropoietin (EPO) from Chinese Hamster Ovary (CHO) cells was increased by reducing agents. Addition of 5 mM cysteamine to serum free medium led to almost 8-fold increase of specific EPO secretion rate although it was not effective in thiol free medium. Each reducing agent has its own optimal concentration for maximizing the EPO secretion from CHO cells.

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

ABSTRACT It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression.

Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO2 addition

The effect of low levels of carbon dioxide (CO2) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO2 addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5–5% CO2 concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO2 supply was highest among the tested cultures. The cell specific secretion rate of EPO (qEPO) of the culture under no CO2 supply was about 3 times higher than that of the culture under 5% CO2 supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO2 environments (air vs. 5% CO2), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO2 environment. Various combinations of the two extreme cases, with 5% CO2 supply (suitable for cell growth) and no CO2 addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (PV) in CHO cells. The PV of the cultures programmed with initial incubation under 5% CO2 followed by no CO2 supply was about 2 times superior to that of the culture incubated only under no CO2 supply. The PV of the culture under no CO2 supply was slightly lower than that of culture grown under 5% CO2. However, the qEPO of the no CO2 supply case was more than 5 times higher than that of the culture under 5% CO2 supply. In conclusion, we have demonstrated that a simple programming of CO2 supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality.

Corrigendum: The Effect of Micro-Spicule Containing Epidermal Growth Factor on Periocular Wrinkles

[This corrects the article on p. 187 in vol. 29, PMID: 28392646.].

The Effect of Micro-Spicule Containing Epidermal Growth Factor on Periocular Wrinkles

Background Micro-needle patches have been recently used to increase skin permeability, which improves drug delivery, and for cosmetic purposes. However, these patches may often have limited efficacy due to insufficient skin penetration and reduced compliance caused by discomfort. Objective We evaluated the efficacy and the safety of soluble micro-spicule containing epidermal growth factor (MS-EGF) for the treatment of periocular wrinkles. Methods Twenty healthy volunteers aged 33 to 54 years were enrolled in a randomized, controlled, split-face study. For 4 weeks, a periocular wrinkle was treated daily with either a soluble MS-EGF cream or a cream containing EGF alone. All subjects underwent 8 weeks of follow-up. Efficacy was assessed using an ultrasonic measurement of dermal depth and density, digital skin image analysis, 5-point photonumeric scale for periocular wrinkles and subjective satisfaction. Results MS-EGF group showed statistically significant increase of dermal depth and density compared to EGF alone group after 4 and 8 weeks. In addition, there was a marked improvement shown in clinical and 3-dimensional skin image in MS-EGF group. The treatments were well-tolerated; no significant side-effect was noted. Conclusion The MS-EGF formulation may represent an effective and biocompatible advance in the treatment of periocular wrinkles.