Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

Phenotypical statin-associated immune-mediated necrotizing myositis with histological features of inclusion body myositis

Introduction:Statin-associated immune-mediated necrotizing myositis(IMNM)is a rare but distinct idiopathic inflammatory myopathy(IIM)that requires early recognition and intervention to prevent irreversible muscle damage.It is typically characterized by active statin use,elevated creatinine kinase(CK)levels,proximal muscle weakness,and at times,a positive 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase(HMGCR)antibody.Treatment includes immediate discontinuation of the statin and may include corticosteroids,intravenous immunoglobulin(IVIG),and/or immunosuppressive therapy.Inclusion body myositis(IBM),another distinct IIM,also presents with elevated CK levels but with insidious onset of distal upper and proximal lower extremity weakness and is typically refractory to treatment.Case Description:A 64-year-old female patient presented with proximal muscle weakness,elevated CK levels,and a positive HMGCR antibody in the setting of statin use with muscle pathology suggestive of both statinassociated IMNM and IBM.She responded to subcutaneous methotrexate and a slow prednisone taper over several months,however,will require close monitoring for symptoms associated with either disease.Conclusion:In conclusion,we report a case of muscle weakness with muscle pathology demonstrating both statin-associated IMNM and IBM.This case highlights the importance of understanding the clinical and pathological features of statin-associated IMNM and IBM.

Effect of Freeze-Thaw and Urea in Solubility of GPC3-Csub Protein Expressed in Escherichia coli

Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.

PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment

Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.

The role of sequestosome 1/p62 protein in amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.

Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl β-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems em- ployed in earlier Src expression.

Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.