Recombinant protein expression: Challenges in production and folding related matters.

Protein folding is a biophysical process by which proteins reach a specific three-dimensional structure. The amino acid sequence of a polypeptide chain contains all the information needed to determine the final three-dimensional structure of a protein. When producing a recombinant protein, several problems can occur, including proteolysis, incorrect folding, formation of inclusion bodies, or protein aggregation, whereby the protein loses its natural structure. To overcome such limitations, several strategies have been developed to address each specific issue. Identification of proper protein refolding conditions can be challenging, and to tackle this high throughput screening for different recombinant protein folding conditions can prove a sound solution. Different approaches have emerged to tackle refolding issues. One particular approach to address folding issues involves molecular chaperones, highly conserved proteins that contribute to proper folding by shielding folding proteins from other proteins that could hinder the process. Proper protein folding is one of the main prerequisites for post-translational modifications. Incorrect folding, if not dealt with, can lead to a buildup of protein misfoldings that damage cells and cause widespread abnormalities. Said post-translational modifications, widespread in eukaryotes, are critical for protein structure, function and biological activity. Incorrect post-translational protein modifications may lead to individual consequences or aggregation of therapeutic proteins. In this review article, we have tried to examine some key aspects of recombinant protein expression. Accordingly, the relevance of these proteins is highlighted, major problems related to the production of recombinant protein and to refolding issues are pinpointed and suggested solutions are presented. An overview of post-translational modification, their biological significance and methods of identification are also provided. Overall, the work is expected to illustrate challenges in recombinant protein expression.

Identification of a novel tailor-made chitinase from white shrimp Fenneropenaeus merguiensis.

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed KM, Vmax and kcat values of 78.18 µM, 0.07261 µM. min-1 and 43.37 s-1, respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste.

Marine chitinolytic enzymes, a biotechnological treasure hidden in the ocean?

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.